Beta-d-2&#39;-deoxy-2&#39;-alpha-fluoro-2&#39;-beta-c-substituted-2-modified-n6-substituted purine nucleotides for hcv treatment

ABSTRACT

A compound of the structure: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt or composition thereof for the treatment of a host infected with or exposed to an HCV virus or other disorders more fully described herein.

PRIORITY

This application is a continuation of U.S. Ser. No. 16/900,397, filedJun. 12, 2020, which is a continuation of U.S. Ser. No. 16/278,621 filedFeb. 18, 2019, which is a continuation U.S. Ser. No. 16/001,549 filedJun. 6, 2018, now U.S. Pat. No. 10,239,911 which is a continuation ofU.S. Ser. No. 15/782,628, now U.S. Pat. No. 10,000,523, filed Oct. 12,2017 which is a continuation of U.S. Ser. No. 15/063,461, now U.S. Pat.No. 9,828,410, filed Mar. 7, 2016 which claims priority to U.S. Ser. No.62/129,319 filed Mar. 6, 2015; U.S. Ser. No. 62/253,958 filed Nov. 11,2015; and, U.S. Ser. No. 62/276,597 filed Jan. 8, 2016. Each of thesereferences is incorporated herewith in their entirety.

FIELD OF THE INVENTION

The present invention is directed to nucleotide compounds andcompositions and uses thereof to treat the Hepatitis C virus (“HCV”).

BACKGROUND OF THE INVENTION

Hepatitis C (HCV) is an RNA single stranded virus and member of theHepacivirus genus. It is estimated that 75% of all cases of liverdisease are caused by HCV. HCV infection can lead to cirrhosis and livercancer, and if left to progress, liver failure which may require a livertransplant. Approximately 170-200 million people worldwide are infected,with an estimated 3-4 million infections in the United States.

RNA polymerase is a key component in the targeting of RNA singlestranded viruses. The HCV non-structural protein NS5B RNA-dependent RNApolymerase is a key enzyme responsible for initiating and catalyzingviral RNA synthesis. As a result, HCV NS5B is an attractive target forthe current drug discovery and development of anti-HCV agents. There aretwo major subclasses of NS5B inhibitors: nucleoside analogs, which areanabolized to their active triphosphates—which act as alternativesubstrates for the polymerase—and non-nucleoside inhibitors (NNIs),which bind to allosteric regions on the protein. Nucleoside ornucleotide inhibitors mimic natural polymerase substrate and act aschain terminators. They inhibit the initiation of RNA transcription andelongation of a nascent RNA chain.

In addition to targeting RNA polymerase, other RNA viral proteins mayalso be targeted in combination therapies. For example, HCV proteinsthat are additional targets for therapeutic approaches are NS3/4A (aserine protease) and NS5A (a non-structural protein that is an essentialcomponent of HCV replicase and exerts a range of effects on cellularpathways).

In December 2013, the first nucleoside NS5B polymerase inhibitorsofosbuvir (Sovaldi®, Gilead Sciences) was approved. Sovaldi® is auridine phosphoramidate prodrug that is taken up by hepatocytes andundergoes intracellular activation to afford the active metabolite;2′-deoxy-2′-α-fluoro-β-C-methyluridine-5′-triphosphate; see structuresbelow:

Sovaldi® is the first drug that has demonstrated safety and efficacy totreat certain types of HCV infection without the need forco-administration of interferon. Sovaldi® is the third drug withbreakthrough therapy designation to receive FDA approval.

In 2014, the U.S. FDA approved Harvoni® (ledispasvir, a NS5A inhibitor,and sofosbuvir) to treat chronic hepatitis C virus genotype 1 infection.Harvoni® is the first combination pill approved to treat chronic HCVgenotype 1 infection. It is also the first approved regimen that doesnot require administration with interferon or ribavirin. In addition,the FDA approved simeprevir (Olysio™) in combination with sofosbuvir(Sovaldi®) as a once-daily, all oral, interferon and ribavirin-freetreatment for adults with genotype 1 HCV infection.

The U.S. FDA also approved AbbVie's VIEKIRA Pak™ in 2014, a multipillpack containing dasabuvir (a non-nucleoside NS5B polymerase inhibitor),ombitasvir (a NS5A inhibitor), paritaprevir (a NS3/4A inhibitor), andritonavir. The VIEKIRA Pak™ can be used with or without the ribavirin totreat genotype 1 HCV infected patients including patients withcompensated cirrhosis. VIEKIRA Pak™ does not require interferonco-therapy.

In July 2015, the U.S. FDA approved Technivie™ and Daklinza™ for thetreatment of HCV genotype 4 and HCV genotype 3 respectively. Technivie™(Ombitasvir/paritaprevir/ritonavir) was approved for use in combinationwith ribavirin for the treatment of HCV genotype 4 in patients withoutscarring and cirrhosis and is the first option for HCV-4 infectedpatients who do not require co-administration with interferon. Daklinza™was approved for use with Sovaldi® to treat HCV genotype 3 infections.Daklinza™ is the first drug that has demonstrated safety and efficacy intreating HCV genotype 3 without the need for co-administration ofinterferon or ribavirin.

In October 2015, the U.S. FDA warned that HCV treatments Viekira Pak andTechnivie can cause serious liver injury primarily in patients withunderlying advanced liver disease, and required that additionalinformation about safety be added to the label.

Other current approved therapies for HCV include interferon alpha-2b orpegylated interferon alpha-2b (Pegintron®), which can be administeredwith ribavirin (Rebetol®), NS3/4A telaprevir (Incivek®, Vertex andJohnson & Johnson), boceprevir (Victrelis™, Merck), simeprevir (Olysio™,Johnson & Johnson), paritaprevir (AbbVie), Ombitasvir (AbbVie), (NNI)Dasabuvir (ABT-333) and Merck's Zepatier™ (a single-tablet combinationof the two drugs grazoprevir and elbasvir).

Additional NS5B polymerase inhibitors are currently under development.Merck is developing the uridine nucleotide prodrug MK-3682 (formerlyIdenix IDX21437). The drug is currently in Phase II combination trials.

United States patents and WO applications which describe nucleosidepolymerase inhibitors for the treatment of Flaviviridae, including HCV,include those filed by Idenix Pharmaceuticals (U.S. Pat. Nos. 6,812,219;6,914,054; 7,105,493; 7,138,376; 7,148,206; 7,157,441; 7,163,929;7,169,766; 7,192,936; 7,365,057; 7,384,924; 7,456,155; 7,547,704;7,582,618; 7,608,597; 7,608,600; 7,625,875; 7,635,689; 7,662,798;7,824,851; 7,902,202; 7,932,240; 7,951,789; 8,193,372; 8,299,038;8,343,937; 8,362,068; 8,507,460; 8,637,475; 8,674,085; 8,680,071;8,691,788, 8,742,101, 8,951,985; 9,109,001; 9,243,025; US2016/0002281;US2013/0064794; WO/2015/095305; WO/2015/081133; WO/2015/061683;WO/2013/177219; WO/2013/039920; WO/2014/137930; WO/2014/052638;WO/2012/154321); Merck (U.S. Pat. Nos. 6,777,395; 7,105,499; 7,125,855;7,202,224; 7,323,449; 7,339,054; 7,534,767; 7,632,821; 7,879,815;8,071,568; 8,148,349; 8,470,834; 8,481,712; 8,541,434; 8,697,694;8,715,638, 9,061,041; 9,156,872 and WO/2013/009737); Emory University(U.S. Pat. Nos. 6,348,587; 6,911,424; 7,307,065; 7,495,006; 7,662,938;7,772,208; 8,114,994; 8,168,583; 8,609,627; US 2014/0212382; andWO2014/1244430); Gilead Sciences/Pharmasset Inc. (U.S. Pat. Nos.7,842,672; 7,973,013; 8,008,264; 8,012,941; 8,012,942; 8,318,682;8,324,179; 8,415,308; 8,455,451; 8,563,530; 8,841,275; 8,853,171;8,871,785; 8,877,733; 8,889,159; 8,906,880; 8,912,321; 8,957,045;8,957,046; 9,045,520; 9,085,573; 9,090,642; and 9,139,604) and (U.S.Pat. Nos. 6,908,924; 6,949,522; 7,094,770; 7,211,570; 7,429,572;7,601,820; 7,638,502; 7,718,790; 7,772,208; RE42,015; U.S. Pat. Nos.7,919,247; 7,964,580; 8,093,380; 8,114,997; 8,173,621; 8,334,270;8,415,322; 8,481,713; 8,492,539; 8,551,973; 8,580,765; 8,618,076;8,629,263; 8,633,309; 8,642,756; 8,716,262; 8,716,263; 8,735,345;8,735,372; 8,735,569; 8,759,510 and 8,765,710); Hoffman La-Roche (U.S.Pat. No. 6,660,721), Roche (U.S. Pat. Nos. 6,784,166; 7,608,599,7,608,601 and 8,071,567); Alios BioPharma Inc. (U.S. Pat. Nos.8,895,723; 8,877,731; 8,871,737, 8,846,896, 8,772,474; 8,980,865;9,012,427; US 2015/0105341; US 2015/0011497; US 2010/0249068;US2012/0070411; WO 2015/054465; WO 2014/209979; WO 2014/100505; WO2014/100498; WO 2013/142159; WO 2013/142157; WO 2013/096680; WO2013/088155; WO 2010/108135), Enanta Pharmaceuticals (U.S. Pat. Nos.8,575,119; 8,846,638; 9,085,599; WO 2013/044030; WO 2012/125900), Biota(U.S. Pat. Nos. 7,268,119; 7,285,658; 7,713,941; 8,119,607; 8,415,309;8,501,699 and 8,802,840), Biocryst Pharmaceuticals (U.S. Pat. Nos.7,388,002; 7,429,571; 7,514,410; 7,560,434; 7,994,139; 8,133,870;8,163,703; 8,242,085 and 8,440,813), Alla Chem, LLC (U.S. Pat. No.8,889,701 and WO 2015/053662), Inhibitex (U.S. Pat. No. 8,759,318 andWO/2012/092484), Janssen Products (U.S. Pat. Nos. 8,399,429; 8,431,588,8,481,510, 8,552,021, 8,933,052; 9,006,29 and 9,012,428) the Universityof Georgia Foundation (U.S. Pat. Nos. 6,348,587; 7,307,065; 7,662,938;8,168,583; 8,673,926, 8,816,074; 8,921,384 and 8,946,244), RFS Pharma,LLC (U.S. Pat. Nos. 8,895,531; 8,859,595; 8,815,829; 8,609,627;7,560,550; US 2014/0066395; US 2014/0235566; US 2010/0279969;WO/2010/091386 and WO 2012/158811) University College CardiffConsultants Limited (WO/2014/076490, WO 2010/081082; WO/2008/062206),Achillion Pharmaceuticals, Inc. (WO/2014/169278 and WO 2014/169280),Cocrystal Pharma, Inc. (U.S. Pat. No. 9,173,893), KatholiekeUniversiteit Leuven (WO 2015/158913), Catabasis (WO 2013/090420) and theRegents of the University of Minnesota (WO 2006/004637).

Nonetheless, there remains a strong medical need to develop anti-HCVtherapies that are safe, effective and well-tolerated. The need isaccentuated by the expectation that drug resistance. More potentdirect-acting antivirals could significantly shorten treatment durationand improve compliance and SVR rates for patients infected with all HCVgenotypes.

It is therefore an object of the present invention to provide compounds,pharmaceutical compositions, and methods and uses to treat and/orprevent infections of HCV.

SUMMARY OF THE INVENTION

It has been discovered that the compounds of Formula I, Formula II,Formula III, Formula IV, Formula V, Formula VI, Formula VII andincluding β-D-2′-deoxy-2′-α-fluoro-2′-β-C-substituted-N⁶-(mono- ordi-methyl) purine nucleotides, are highly active against the HCV viruswhen administered in an effective amount to a host in need thereof. Thehost can be a human or any animal that carries the viral infection.

Disclosed nucleotides include those with nanomolar activity against HCVin vitro and therapeutic indices that range to 25,000 or more.

Surprisingly, the parent N⁶-(methyl) purine nucleosides of disclosedcompounds had not been developed or specifically disclosed as drugcandidates prior to this invention. For example, it was reported in 2010that 3′-azido-N⁶-dimethyl-2,6-diaminopurine is not substantiallydeaminated by adenosine deaminase over a long period (120 minutes), andfor that reason it had been considered an inappropriate compound toderivatize as a drug (see for example, WO 2010/091386, page 86 andcorresponding U.S. Pat. No. 8,609,627).

However, it has now been discovered that compounds of the presentinvention are anabolized to a 5-monophosphate of theN⁶-substituted-purine without substantial N⁶-deamination and thensubsequently anabolized at the 6-position to generate active guaninetriphosphate compounds, in a manner that provides exceptional activityand therapeutic index.

In particular, it has been discovered that a 5′-stabilized phosphateprodrug or derivative ofβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diaminopurinenucleotide, as well asβ-2′-deoxy-2′-α-fluoro-2′-β-methyl-N-dimethyl-2,6-diaminopurinenucleotide, and otherβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotides as described below, are highly active against HCV. This issurprising because the activity of the parent nucleosideβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diaminopurine in areplicon assay (EC₅₀=15.7 micromolar) indicates that it is not suitablefor use as a human drug due to insufficient activity (in combinationwith the reference WO 2010/091386, page 86 and corresponding U.S. Pat.No. 8,609,627 that suggests that N⁶-methyl-2,6-diaminopurines are notdeaminated in vivo) however, the stabilized racemic phosphate prodrug(phosphoramidate) exhibits an EC₅₀=26 nanomolar (nM), in a repliconassay, which is at least an 600 fold increase in activity. Thecorresponding (S)-phosphoramidate exhibits an EC₅₀=4 nM, which is atleast a 3,900 fold increase in activity; see the structure below andcompound 5-2 in Table 7. With a TC₅₀ greater than one hundredmicromolar, the compound thus has a therapeutic index of greater than25,000. For comparison, Sofosbuvir has an EC₅₀=53 nM, a TC₅₀ greaterthan one hundred micromolar and a therapeutic index greater than 1,920.

Likewise, the activity of the parent nucleosideβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-dimethyl-2,6-diaminopurine in areplicon assay (EC₅₀=10.7 micromolar, “M”) indicates that it is also notsuitable for use as a human drug due to insufficient activity, however,the stabilized racemic phosphate prodrug (phosphoramidate) exhibits anEC₅₀=12 nM, in a replicon assay, which is more than a 890 fold increasein activity. The corresponding (S)-phosphoramidate (compound 25, Table7) also exhibits an EC₅₀=4 nM, which is at least a 2,600 fold increasein activity; see the structure below. In addition, compound 25 also hasa therapeutic index of greater than 25,000.

In another example, the compound isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-N-cyclopropyl-amino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninateexhibited an EC₅₀=7 nM and the corresponding (S)-phosphoramidateexhibited an EC₅₀=5 nM in a replicon assay; see compound 27 in Table 7and the structure below.

As stated above, the metabolism of theβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diaminopurinenucleoside as a phosphoramidate involves the production of a5′-monophosphate and the subsequent anabolism of theN⁶-methyl-2,6-diaminopurine base to generate theβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-guanine nucleoside as the5′-monophosphate. The monophosphate is then further anabolized to theactive species; the 5′-triphosphate. Theβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-guanine triphosphate has anIC₅₀=0.15 μM against the HCV genotype 1b NS5B polymerase.

Thus, in one embodiment, the invention is:

wherein:

-   -   Y is NR¹R²    -   R¹ is C₁-C₅alkyl (including methyl, ethyl, n-propyl, iso-propyl,        n-butyl, iso-butyl, sec-butyl, tert-butyl and pentyl),        C₁-C₅haloalkyl (including CH₂F, CHF₂, CF₃, CH₂CF₃, CF₂CH₃ and        CF₂CF₃), C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(heterocycle),        —(C₀-C₂alkyl)(aryl), —(C₀-C₂alkyl)(heteroaryl), —OR²⁵,        —C(O)R^(3C) (including —C(O)CH₃, —C(O)CH₂CH₃—C(O)CH(CH₃)₂,        —C(O)OCH₃, —C(O)OC₂H₅, —C(O)OC₃H₇, —C(O)OC₄H₉, and —C(O)OC₅H₁₁),        —C(S)R^(3D), or —SO₂R²⁸ each of which can be optionally        substituted;    -   R² is hydrogen, C₁-C₅alkyl (including methyl, ethyl, n-propyl,        iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl and        pentyl), C₁-C₅haloalkyl (including CHF₂, CHF₂, CF₃, CH₂CF₃ and        CF₂CF₃), —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —C(O)R^(3C) (including        —C(O)CH₃, —C(O)CH₂CH₃—C(O)CH(CH₃)₂, —C(O)OCH₃, —C(O)OC₂H,        —C(O)OC₃H₇, —C(O)OC₄H₉, and —C(O)OC₅H₁₁), —(C₀-C₂alkyl)(aryl),        —(C₀-C₂alkyl)(heterocycle), —(C₀-C₂alkyl)(heteroaryl); and        wherein at least one of R¹ and R² is methyl, CH₂F, CHF₂ or CF₃;    -   R³ is hydrogen,

diphosphate, triphosphate, an optionally substituted carbonyl linkedamino acid, or —C(O)R^(3C);R^(3A) can be selected from O⁻, OH, an —O-optionally substituted aryl,an —O-optionally substituted heteroaryl, or an optionally substitutedheterocyclyl;

-   -   R^(3B) can be selected from O⁻, OH, an optionally substituted        N-linked amino acid or an optionally substituted N-linked amino        acid ester;        R^(3C) is alkyl, alkenyl, alkynyl, —(C₀-C₂)(cycloalkyl),        —(C₀-C₂)(heterocyclo), —(C₀-C₂)(aryl), —(C₀-C₂)(heteroaryl),        —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(C₀-C₂)(cycloalkyl),        —O—(C₀-C₂)(heterocyclo), —O—(C₀-C₂)(aryl), or        —O—(C₀-C₂)(heteroaryl), each of which can be optionally        substituted;    -   R⁴ is a monophosphate, diphosphate, triphosphate, or a        stabilized phosphate prodrug, including but not limited to a        phosphoramidate, a thiophosphoramidate, or any other moiety that        is metabolized to a monophosphate, diphosphate or triphosphate        in vivo in the host human or animal; or    -   R³ and R⁴ together with the oxygens that they are bonded to can        form a 3′,5′-cyclic prodrug, including but not limited to, a        3′,5′-cyclic phosphate prodrug;    -   R¹² is CH₃, CH₂F, CHF₂, CF₃, or ethynyl.

In one embodiment, the invention is:

wherein:

-   -   Y is NR¹R²;    -   R¹ is C₁-C₅alkyl (including methyl, ethyl, n-propyl, iso-propyl,        n-butyl, iso-butyl, sec-butyl, tert-butyl and pentyl),        C₁-C₅haloalkyl (including CH₂F, CHF₂, CF₃, CH₂CF₃, CF₂CH₃ and        CF₂CF₃), C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(heterocycle),        —(C₀-C₂alkyl)(aryl), —(C₀-C₂alkyl)(heteroaryl), —OR²⁵,        —C(O)R^(3C) (including —C(O)CH₃, —C(O)CH₂CH₃—C(O)CH(CH₃)₂,        —C(O)OCH₃, —C(O)OC₂H₅, —C(O)OC₃H₇, —C(O)OC₄H₉, and —C(O)OC₅H₁₁),        —C(S)R^(3D), or —SO₂R²⁸ each of which can be optionally        substituted;    -   R² is hydrogen, optionally substituted C₁-C₅alkyl (including        methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,        sec-butyl, tert-butyl and pentyl), C₁-C₅haloalkyl (including        CHF₂, CHF₂, CF₃, CH₂CF₃ and CF₂CF₃), optionally substituted        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), optionally substituted        —(C₀-C₂alkyl)(heterocycle), optionally substituted        —(C₀-C₂alkyl)(aryl), optionally substituted        —(C₀-C₂alkyl)(heteroaryl), —C(O)R^(3C) (including —C(O)CH₃,        —C(O)CH₂CH₃—C(O)CH(CH₃)₂, —C(O)OCH₃, —C(O)OC₂H₅, —C(O)OC₃H₇,        —C(O)OC₄H₉, and —C(O)OC₅H₁₁), —C(S)R^(3D), or —SO₂R²⁸; and        wherein at least one of R¹ and R² is methyl, CH₂F, CHF₂ or CF₃;    -   R³ is hydrogen,

diphosphate, triphosphate, an optionally substituted carbonyl linkedamino acid, or —C(O)R^(3C);

-   -   R^(3A) can be selected from O⁻, OH, an —O-optionally substituted        aryl, an —O-optionally substituted heteroaryl, or an optionally        substituted heterocyclyl;    -   R^(3B) can be selected from O⁻, OH, an optionally substituted        N-linked amino acid or an optionally substituted N-linked amino        acid ester;    -   R^(3C) is alkyl, alkenyl, alkynyl, —(C₀-C₂)(cycloalkyl),        —(C₀-C₂)(heterocyclo), —(C₀-C₂)(aryl), —(C₀-C₂)(heteroaryl),        —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(C₀-C₂)(cycloalkyl),        —O—(C₀-C₂)(heterocyclo), —O—(C₀-C₂)(aryl),        —O—(C₀-C₂)(heteroaryl), —S-alkyl, —S-alkenyl, —S-alkynyl,        —S—(C₀-C₂)(cycloalkyl), —S—(C₀-C₂)(heterocycle),        —S—(C₀-C₂)(aryl), or —S—(C₀-C₂)(heteroaryl) each of which can be        optionally substituted;    -   R^(3D) is alkyl, alkenyl, alkynyl, —(C₀-C₂)(cycloalkyl),        —(C₀-C₂)(heterocyclo), —(C₀-C₂)(aryl), —(C₀-C₂)(heteroaryl),        —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(C₀-C₂)(cycloalkyl),        —O—(C₀-C₂)(heterocyclo), —O—(C₀-C₂)(aryl), or        —O—(C₀-C₂)(heteroaryl), each of which can be optionally        substituted;    -   R⁴ is a monophosphate, diphosphate, triphosphate, or a        stabilized phosphate prodrug, including but not limited to a        phosphoramidate, a thiophosphoramidate, or any other moiety that        is metabolized to a monophosphate, diphosphate or triphosphate        in vivo in the host human or animal; or    -   R³ and R⁴ together with the oxygens that they are bonded to can        form a 3′,5′-cyclic prodrug, including but not limited to, a        3′,5′-cyclic phosphate prodrug;    -   R⁵ is C₁-C₅alkyl (including methyl, ethyl, n-propyl, iso-propyl,        n-butyl, iso-butyl, sec-butyl, tert-butyl and pentyl),        C₁-C₅haloalkyl (including CHF₂, CHF₂, CF₃, CH₂CF₃ and CF₂CF₃),        C₂-C₆ alkenyl, C₂-C₆ alkynyl, —(C₀-C₂alkyl)(C₃-C₆cycloalkyl),        —(C₀-C₂alkyl)(heterocycle), —(C₀-C₂alkyl)(aryl),        —(C₀-C₂alkyl)(heteroaryl), —OR²⁵, —C(O)R^(3C) (including        —C(O)CH₃, —C(O)CH₂CH₃—C(O)CH(CH₃)₂, —C(O)OCH₃, —C(O)OC₂H,        —C(O)OC₃H₇, —C(O)OC₄H₉, and —C(O)OC₅H₁₁), —C(S)R^(3D), or        —SO₂R²⁸ each of which can be optionally substituted;    -   R⁶ is hydrogen, optionally substituted C₁-C₅alkyl (including        methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,        sec-butyl, tert-butyl and pentyl), C₁-C₅haloalkyl (including        CHF₂, CHF₂, CF₃, CH₂CF₃ and CF₂CF₃), optionally substituted        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), optionally substituted        —(C₀-C₂alkyl)(heterocycle), optionally substituted        —(C₀-C₂alkyl)(aryl), optionally substituted        —(C₀-C₂alkyl)(heteroaryl), —C(O)R^(3C) (including —C(O)CH₃,        —C(O)CH₂CH₃—C(O)CH(CH₃)₂, —C(O)OCH₃, —C(O)OC₂H, —C(O)OC₃H₇,        —C(O)OC₄H₉, and —C(O)OC₅H₁₁), —C(S)R^(3D), or —SO₂R²⁸; or    -   R⁵ and R⁶ together with the nitrogen that they are bonded to can        form a heterocyclic ring;    -   R¹² is CH₃, CH₂F, CHF₂, CF₃, or ethynyl;    -   R²² is Cl, Br, F, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, —(C₁-C₂alkyl)(C₃-C₆cycloalkyl),        —(C₀-C₂alkyl)(C₃-C₆heterocycle), —(C₀-C₂alkyl)(aryl),        —(C₀-C₂alkyl)(heteroaryl); —ONHC(═O)OR²³, —NHOR²⁴, —OR²⁵, —SR²⁵,        —NH(CH₂)₁₋₄N(R²⁶)₂, —NHNHR²⁶, —N═NR²⁷, —NHC(O)NHNHR²⁷,        —NHC(S)NHNHR²⁷, —C(O)NHNHR²⁷, —NR²⁷SO₂R²⁸, —SO₂NR²⁷R²⁹,        —C(O)NR²⁷R²⁹, —CO₂R²⁹, —SO₂R²⁹,

—P(O)H(OR²⁹), —P(O)(OR²⁹)(OR³⁰), —P(O)(OR²⁹)(NR²⁹R³⁰) or —NR⁵R⁶;

-   -   for example including but not limited to the following        embodiments, chloro, bromo, fluoro, cyano, azido, ethyl,        n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl        and n-pentyl, 1,1-dimethylpropyl, 2,2-dimtheylpropyl,        3-methylbutyl, 1-methylbutyl, 1-ethylpropyl, vinyl, allyl,        1-butynyl, 2-butynyl, acetylenyl, cyclopropyl, cyclobutyl,        cyclopentyl, cyclohexyl, —(CH₂)-cyclopropyl, —(CH₂)-cyclobutyl,        —(CH₂)-cyclopentyl, —(CH₂)-cyclohexyl, aziridine, oxirane,        thiirane, azetidine, oxetane, thietane, pyrrolidine,        tetrahydrofuran, thiolane, pyrazolidine, piperidine, oxane,        thiane, —(CH₂)-aziridine, —(CH₂)-oxirane, —(CH₂)-thiirane,        —(CH₂)-azetidine, —(CH₂)-oxetane, —(CH₂)-thietane,        —(CH₂)-pyrrolidine, —(CH₂)-tetrahydrofuran, —(CH₂)-thiolane,        —(CH₂)-pyrazolidine, —(CH₂)-piperidine, —(CH₂)-oxane,        —(CH₂)-thiane, phenyl, pyridyl, —ONHC(═O)OCH₃, —ONHC(═O)OCH₂CH₃,        —NHOH, NHOCH₃, —OCH₃, OC₂H₅, —OPh, OCH₂Ph, —SCH₃, —SC₂H₅, —SPh,        SCH₂Ph, —NH(CH₂)₂NH₂, —NH(CH₂)₂N(CH₃)₂, —NHNH₂, —NHNHCH₃, —N═NH,        —N═NCH₃, —N═NCH₂CH₃, —NHC(O)NHNH₂, —NHC(S)NHNH₂, —C(O)NHNH₂,        —NHSO₂CH₃, —NHSO₂CH₂CH₃, —SO₂NHCH₃, —SO₂N(CH₃)₂, —C(O)NH₂,        —C(O)NHCH₃, —C(O)N(CH₃)₂, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂Ph,        —CO₂CH₂Ph, —SO₂CH₃, —SO₂CH₂CH₃, —SO₂Ph, —SO₂CH₂Ph,

—P(O)H(OH), —P(O)H(OCH₃), —P(O)(OH)(OH), —P(O)(OH)(OCH₃),—P(O)(OCH₃)(OCH₃), —P(O)(OH)(NH₂), —P(O)(OH)(NHCH₃), —P(O)(OH)N(CH₃)₂,—NHC(O)CH₃, —NHC(O)CH₂CH₃, —NHC(O)CH(CH₃)₂, —NHC(O)OCH₃, —NHC(O)OCH₂CH₃,—NHC(O)OCH(CH₃)₂, —NHC(O)OCH₂CH₂CH₃, —NHC(O)OCH₂CH₂CH₂CH₃ and—NHC(O)OCH₂CH₂CH₂CH₂CH₃;

-   -   R²³ is C₁-C₅alkyl, —(C₀-C₂alkyl)(C₃-C₆cycloalkyl),        —(C₀-C₂alkyl)(heterocycle)-(C₀₋₂ alkyl)(aryl) or        —(C₀-C₂alkyl)(heteroaryl) each of which can be optionally        substituted;    -   R²⁴ is hydrogen, C₁-C₆ alkyl, —(C₀-C₂alkyl)(C₃-C₆cycloalkyl),        —(C₁-C₂alkyl)(C₃-C₆heterocycle) —(C₀-C₂alkyl)(aryl) or        —(C₀-C₂alkyl)(heteroaryl) wherein except for the hydrogen each        of which can be optionally substituted;    -   R²⁵ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(C₃-C₆heterocycle),        —(C₀-C₂alkyl)(aryl) or —(C₀-C₂alkyl)(heteroaryl) wherein except        for the hydrogen each of which can be optionally substituted;    -   R²⁶ is independently selected from hydrogen, C₁-C₆alkyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(heterocycle),        —(C₀-C₂alkyl)(aryl), or —(C₀-C₂alkyl)(heteroaryl) wherein except        for the hydrogen each of which can be optionally substituted;    -   R²⁷ hydrogen or optionally substituted C₁-C₆ alkyl;    -   R²⁸ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(C₃-C₆heterocycle),        —(C₀-C₂alkyl)(aryl) or —(C₀-C₂alkyl)(heteroaryl) each of which        can be optionally substituted;    -   R²⁹ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(C₃-C₆heterocycle),        —(C₀-C₂alkyl)(aryl) or —(C₀-C₂alkyl)(heteroaryl) wherein except        for the hydrogen each of which can be optionally substituted; or    -   R²⁷ and R²⁹ together with the nitrogen that they are bonded to        can form a heterocyclic ring;    -   R³⁰ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃C₆cycloalkyl), —(C₀-C₂alkyl)(C₃-C₆heterocycle),        —(C₀-C₂alkyl)(aryl) or —(C₀-C₂alkyl)(heteroaryl) wherein except        for the hydrogen each of which can be optionally substituted; or    -   R²⁹ and R³⁰ can be bonded together to form a heterocyclic ring;    -   x is 1, 2 or 3.

The metabolism of theβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-dimethyl-2,6-diaminopurinenucleotide involves both the formation of theβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-dimethyl-2,6-diaminopurinenucleoside triphosphate as well as the generation of the correspondingguanine nucleoside triphosphate. See Scheme 2 and 3.

2′-Deoxy-2′-α-fluoro-2′-β-C-substituted-N⁶-substituted-2,6-diaminopurinenucleotides can be further substituted at the N²-position by alkylationor acylated which can modify the lipophilicity, pharmacokinetics and/ortargeting of the nucleotide to the liver. It has been discovered that2′-deoxy-2′-α-fluoro-2′-β-C-substituted-N⁶-substituted-2,6-diaminopurinenucleotides modified at the 2-position of the diaminopurine can bedealkylated or deacylated by hepatic enzymes to further increase thespecificity of the nucleotide derivatives both in vitro and in vivo,unless the N²-amino group is completely replaced by a different moiety,as described herein, such as fluoro. For example, the nucleosidephosphoramidate2′-deoxy-2′-α-fluoro-2′-β-methyl-N²-methyl-N⁶-methyl-2,6-diaminopurinenucleoside phosphoramidate is dealkylated to2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diaminopurine nucleosidephosphoramidate when incubated with a human liver S9 fraction in vitro,up to 60 minutes, these conditions mimics in vivo conditions. In oneembodiment, N² modifications will increase cell permeability andhepatitic targeting.

Despite the volume of antiviral nucleoside literature and patentfilings, the 5′-stabilized phosphate derivative of2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diaminopurine nucleoside,2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-dimethyl-2,6-diaminopurinenucleoside, and otherβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleoside derivatives as described herein have not been specificallydisclosed, nor have their advantageous activities been described.

Unless otherwise specified, the compounds described herein are providedin the β-D-configuration. Likewise, when in phosphoramide orthiophosphoramidate form, the amino acid portion can be in the L- orD-configuration. In an alternative embodiment, the compounds can beprovided in a β-L-configuration. Likewise, any substituent group thatexhibits chirality can be provided in racemic, enantiomeric,diastereomeric form or any mixture thereof. Where a phosphoramidate,thiophosphoramidate or other stabilized phosphorus prodrug in which thephosphorus exhibits chirality is used as the R⁴ stabilized phosphateprodrug, it can be provided as an R or S chiral phosphorus derivative ora mixture thereof, including a racemic mixture. All of the combinationsof these stereoconfigurations are included in the invention describedherein.

Accordingly, the present invention includes a compound of Formula I-VII,or a pharmaceutically acceptable composition, salt, or prodrug thereof,as described herein:

In one specific embodiment, the parent nucleoside, i.e., the nucleosidewherein R⁴ is hydrogen and the 5′-position thus has a hydroxyl group, isnot substantially deaminated by adenosine deaminase under conditionsthat mimic the in vivo environment (e.g., ambient temperature andaqueous physiological pH), for a period of 7 minutes, 10 minutes, 30minutes, 60 minutes or 120 minutes. Unless otherwise stated, the timeperiod is 30 minutes. In this embodiment, the term “not substantiallydeaminated” means that the parent compound is not converted to thecorresponding guanine derivative, or 6-oxo derivative, in an amountsufficient to provide a therapeutic effect in vivo.

Compounds, methods, and compositions are provided for the treatment of ahost infected with a HCV virus via administration of an effective amountof the compound or its pharmaceutically acceptable salt.

The compounds and compositions can also be used to treat relatedconditions such as anti-HCV antibody positive and antigen positiveconditions, viral-based chronic liver inflammation, liver cancerresulting from advanced hepatitis C, cirrhosis, chronic or acutehepatitis C, fulminant hepatitis C, chronic persistent hepatitis C andanti-HCV-based fatigue.

The compound or formulations that include the compounds can also be usedprophylactically to prevent or restrict the progression of clinicalillness in individuals who are anti-HCV antibody or antigen positive orwho have been exposed to hepatitis C.

In another embodiment, compounds of Formula Ia are disclosed:

-   -   wherein.        Y, R³ and R⁴ are as defined above.

In one embodiment of Formula Ia, R³ is hydrogen.

In one embodiment of Formula Ia, when Y is NR¹R², R¹ is methyl and R² ishydrogen.

In one embodiment of Formula Ia, when Y is NR¹R², both R¹ and R² aremethyl.

In one embodiment of Formula Ia, when Y is NR¹R², R¹ is methyl and R² iscyclopropyl.

In another embodiment, compounds of Formula Ib are disclosed:

-   -   wherein:    -   Y, R³ and R⁴ are as defined above.

In one embodiment of Formula Ib, R³ is hydrogen.

In one embodiment of Formula Ib, when Y is NR¹R², R¹ is methyl and R² ishydrogen.

In one embodiment of Formula Ib, when Y is NR¹R², both R¹ and R² aremethyl.

In one embodiment, compounds of Formula II are disclosed:

-   -   wherein:    -   Y, R³, R⁴, R¹² and R²² are as defined above.

In another embodiment, compounds of Formula IIa are disclosed:

-   -   wherein:    -   Y, R³, R⁴ and R²² are as defined above.

In another embodiment, compounds of Formula IIb are disclosed:

-   -   wherein:    -   Y, R³, R⁴, and R²² are as defined above.

In one embodiment, compounds of Formula III are disclosed:

-   -   wherein the variables Y, R³, R⁷, R⁸, R^(9a), R^(9b), R¹⁰, R¹²        and R²² are described herein.

In one embodiment, compounds of Formula IV are disclosed:

-   -   wherein the variables Y, R³, R⁷, R⁸, R^(9a), R^(9b), R¹⁰ and R²²        are described herein.

In one embodiment, compounds of Formula V are disclosed:

-   -   wherein the variables Y, R³, R⁷, R⁸, R^(9a), R^(9b), R¹⁰ and R²²        are described herein. In one embodiment, compounds of Formula VI        are disclosed:

-   -   wherein:    -   R⁴¹ is halogen (in particular F or Cl), OR³, N₃, NH₂ or CN; and    -   the variables Y, R³, R⁴, and R¹² are described herein.

In one embodiment, compounds of Formula VII are disclosed:

Wherein the variables Y, R³, R⁴, R¹² and R⁴¹ are described herein.

The phosphorus in any of the Formulas above may be chiral and thus canbe provided as an R or S enantiomer or mixture thereof, including aracemic mixture.

Compound 5 was separated into the enantiomer compounds 5-1 and 5-2.Compound 5-2 was also prepared by chiral synthesis and assigned compound24.

In one embodiment, compounds, methods, and compositions are provided forthe treatment of a host infected with or exposed to hepatitis Cdescribed herein. The compounds of the invention can be administered inan effective amount alone or in combination with another anti-HCV drug,to treat the infected host. In certain embodiments, it is useful toadminister a combination of drugs that modulates the same or a differentpathway or inhibits a different target in the virus. As the disclosedβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotides are NS5B polymerase inhibitors, it may be useful toadminister the compound to a host in combination with a proteaseinhibitor, such as an NS3/4A protease inhibitor (for example, telaprevir(Incivek®) boceprevir (Victrelis™) simeprevir (Olysio™), orparitaprevir, or an NS5A inhibitor (for example, Ombitasvir). Thecompounds of the invention can also be administered in combination witha structurally different NS5B polymerase inhibitor such as anothercompound described herein or below, including Gilead's Sovaldi®. Thecompounds of the invention can also be administered in combination withinterferon alfa-2a, which may be pegylated or otherwise modified, and/orribavirin.

The β-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substitutedpurine nucleotides of the invention are typically administered orally,for example in pill or tablet form, but may be administered via an otherroute which the attending physician considers appropriate, including viaintravenous, transdermal, subcutaneous, topical, parenteral, or othersuitable route.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a sample chromatogram of a semi-prep run illustrating theseparation of the stereoisomers of Compound 5 using a Phenominex Lunacolumn as disclosed in Example 9. The y axis is shown in mAU and the xaxis is measured in minutes.

FIG. 2 is a graph of the HCV replication inhibition curves for Compound5-2 (Table 7) and Sofosbuvir. Compound 5-2 has an EC₅₀=4 nM, a TC₅₀greater than one hundred micromolar and a therapeutic index of greaterthan 25,000. Sofosbuvir has an EC₅₀=53 nM, a TC₅₀ greater than onehundred micromolar and a therapeutic index greater than 1,920. They-axis is the percent of virus control and the x-axis is theconcentration of drug in M.

FIG. 3 is a graph of the HCV replication inhibition curves for Compound25 (Table 7) and Sofosbuvir. As described in Example 27, Compound 25 hasan EC₅₀=4 nM, a TC₅₀ of greater than 100 μM, and a therapeutic index ofgreater than 25,000. Sofosbuvir has an EC₅₀=53 nM, a TC₅₀ greater thanone hundred micromolar and a therapeutic index greater than 1,920. They-axis is the percent of virus control and the x-axis is theconcentration of drug in μM.

FIG. 4 is an intra-assay comparison of the anti-HCV activity forCompounds 5-2, 25, 27 (Table 7) and Sofosbuvir. The y-axis is thepercent of virus control and the x-axis is the concentration of drug inM. See, Example 27.

FIG. 5 is a graph that shows the stability of compounds 5-2; theN²-acetate of compound 5-2, the N²-butyrate of compound 5-2; theN²-methyl derivative of compound 5-2; and the N²-n-pentylcarbamate ofcompound 5-2 in human blood. The x axis is incubation time measured inminutes and the y axis is the measurement of the percent of the parentcompound remaining.

FIG. 6 is a graph showing the in vitro time course dealkylation of2′-deoxy-2′-α-fluoro-2′-β-methyl-N²-methyl-N⁶-methyl-2,6-diaminopurinenucleoside phosphoramidate to2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diaminopurine nucleosidephosphoramidate in the presence of a human liver S9 fraction. The x axisis measured in minutes and the y axis is the measurement of theconcentration of the compound remaining in nM.

FIG. 7 is a graph showing the stability of compounds 5-2; the N²-acetateof compound 5-2, the N²-butyrate of compound 5-2; the N²-methylderivative of compound 5-2; and the N²-n-pentylcarbamate of compound 5-2in the presence of a human liver S9 fraction. The x axis is measured inminutes and the y axis is the measurement of percent compound remaining.

FIG. 8 shows the predominant Compound 25 metabolites generated in humanhepatocytes. The x axis is incubation time in hours. The y axis isintracellular concentration in μmol/10⁶ cells. See Example 33.

FIG. 9 shows the predominant Compound 27 metabolites generated in humanhepatocytes. The x axis is incubation time in hours. The y axis isintracellular concentration in μmol/10⁶ cells. See Example 33.

FIG. 10 shows the predominant Compound 5-2 metabolites generated inhuman hepatocytes. The x axis is incubation time in hours. The y axis isintracellular concentration in μmol/10⁶ cells. See Example 33.

FIG. 11 is a graph showing the activation pathways for Compounds 25, 27and 5-2. As can be seen, Compounds 25, 27 and 5-2 are converted to theircorresponding monophosphate analogs which are subsequently metabolizedto a common MP analog; β-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-guaninemonophosphate. The monophosphate is then stepwise phosphorylated to theactive triphosphate: β-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-guaninetriphosphate. See Example 33.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein is a compound, method, and compositionfor the treatment of infections in or exposure to humans and other hostanimals of the HCV virus that includes the administration of aneffective amount of a compound of Formula I-VII as described herein or apharmaceutically acceptable salt or prodrug thereof, optionally in apharmaceutically acceptable carrier. The compounds of this inventioneither possess antiviral activity, or are metabolized to a compound thatexhibits such activity.

The compounds and compositions can also be used to treat conditionsrelated to or occurring as a result of a HCV viral exposure. Forexample, the active compound can be used to treat HCV antibody positiveand HCV antigen positive conditions, viral-based chronic liverinflammation, liver cancer resulting from advanced hepatitis C,cirrhosis, acute hepatitis C, fulminant hepatitis C, chronic persistenthepatitis C, and anti-HCV-based fatigue. In one embodiment, thecompounds or formulations that include the compounds can also be usedprophylactically to prevent or retard the progression of clinicalillness in individuals who are HCV antibody or HCV antigen positive orwho have been exposed to hepatitis C.

In particular, it has been discovered that a 5′-stabilized phosphateprodrug or derivative ofβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diamino purinenucleotide, as well asβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-dimethyl-2,6-diamino purinenucleotide, and otherβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotides as described below, are highly active against HCV. This issurprising because the activity of the parent nucleosideβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diamino purine in areplicon assay (EC₅₀=15.7 micromolar) indicates that it is not suitablefor use as a human drug due to insufficient activity, however, thestabilized phosphate prodrug (phosphoramidate) exhibits an EC₅₀=26nanomolar, in a replicon assay, which is at least an 870 fold increasein activity. Likewise, the activity of the parent nucleosideβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-dimethyl-2,6-diaminopurine in areplicon assay (EC₅₀=10.7 micromolar, “M”) indicates that it is also notsuitable for use as a human drug due to insufficient activity, however,the stabilized phosphate prodrug (phosphoramidate) exhibits an EC₅₀=12nanomolar, (“nM”), in a replicon assay, which is more than a 1,300 foldincrease in activity.

Despite the volume of antiviral nucleoside literature and patentfilings, the 5′-stabilized phosphate derivative of2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diamino purinenucleotide, 2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-dimethyl-2,6-diaminopurine nucleotide, and otherβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotides have not been specifically disclosed.

Unless otherwise specified, the compounds described herein are providedin the β-D-configuration. In an alternative embodiment, the compoundscan be provided in a β-L-configuration. Likewise, any substituent groupthat exhibits chirality can be provided in racemic, enantiomeric,diastereomeric form or any mixture thereof. Where a phosphoramidate,thiophosphoramidate or other stabilized phosphorus prodrug in which thephosphorus exhibits chirality is used as the R⁴ stabilized phosphateprodrug, it can be provided as an R or S chiral phosphorus derivative ora mixture thereof, including a racemic mixture. The amino acid of thephosphoramidate or thiophosphoramidate can be in the D- orL-configuration, or a mixture thereof, including a racemic mixture. Allof the combinations of these stereo configurations are included in theinvention described herein.

The present invention includes the following features:

-   -   (a) a compound of Formula I-VII as described herein, and        pharmaceutically acceptable salts and prodrugs thereof,    -   (b) Formulas I-VII as described herein, and pharmaceutically        acceptable salts and prodrugs thereof for use in the treatment        or prophylaxis of a hepatitis C virus infection;    -   (c) use of Formulas I-VII, and pharmaceutically acceptable salts        and prodrugs thereof in the manufacture of a medicament for        treatment of a hepatitis C virus infection;    -   (d) a method for manufacturing a medicament intended for the        therapeutic use for treating a hepatitis C virus infection,        characterized in that a Formulas I-VII as described herein is        used in the manufacture;    -   (e) a pharmaceutical formulation comprising an effective        host-treating amount of the Formulas I-VII or a pharmaceutically        acceptable salt or prodrug thereof together with a        pharmaceutically acceptable carrier or diluent;    -   (f) Formulas I-VII as described herein substantially in the        absence of stereoisomers of the described compound, or        substantially isolated from other chemical entities; and,    -   (g) processes for the preparation of therapeutic products that        contain an effective amount of a Formulas I-VII, as described        herein.

I. 2′-Deoxy-2′-α-Fluoro-2′-β-C-Substituted-2-Modified-N⁶-SubstitutedPurine Nucleotides of the Invention

The active compounds of the invention are those depicted, for example,in Formula I, which can be provided in a pharmaceutically acceptablecomposition, salt or prodrug thereof:

-   -   wherein:    -   Y is NR¹R²;    -   R¹ is C₁-C₅alkyl (including methyl, ethyl, n-propyl, iso-propyl,        n-butyl, iso-butyl, sec-butyl, tert-butyl and pentyl),        C₁-C₅haloalkyl (including CH₂F, CH₂F, CF₃, CH₂CF₃, CF₂CH₃ and        CF₂CF₃), C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(heterocycle),        —(C₀-C₂alkyl)(aryl), —(C₀-C₂alkyl)(heteroaryl), —OR²⁵,        —C(O)R^(3C) (including —C(O)CH₃, —C(O)CH₂CH₃—C(O)CH(CH₃)₂,        —C(O)OCH₃, —C(O)OC₂H₅, —C(O)OC₃H₇, —C(O)OC₄H₉, and —C(O)OC₅H₁₁),        —C(S)R^(3D), or —SO₂R²⁸ each of which can be optionally        substituted;    -   R² is hydrogen, optionally substituted C₁-C₅alkyl (including        methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,        sec-butyl, tert-butyl and pentyl), C₁-C₅haloalkyl (including        CHF₂, CH₂F, CF₃, CH₂CF₃ and CF₂CF₃), optionally substituted        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), optionally substituted        —(C₀-C₂alkyl)(heterocycle), optionally substituted        —(C₀-C₂alkyl)(aryl), optionally substituted        —(C₀-C₂alkyl)(heteroaryl), —C(O)R^(3C) (including —C(O)CH₃,        —C(O)CH₂CH₃—C(O)CH(CH₃)₂, —C(O)OCH₃, —C(O)OC₂H₅, —C(O)OC₃H₇,        —C(O)OC₄H₉, and —C(O)OC₅H₁₁), —C(S)R^(3D), or —SO₂R²⁸; and        wherein at least one of R¹ and R² is methyl, CH₂F, CHF₂ or CF₃;    -   R³ is hydrogen,

-   -   diphosphate, triphosphate, an optionally substituted carbonyl        linked amino acid, or —C(O)R^(3C);    -   R^(3A) can be selected from O⁻, OH, an —O-optionally substituted        aryl, an —O-optionally substituted heteroaryl, or an optionally        substituted heterocyclyl;    -   R^(3B) can be selected from O⁻, OH, an optionally substituted        N-linked amino acid or an optionally substituted N-linked amino        acid ester;    -   R^(3C) is alkyl, alkenyl, alkynyl, —(C₀-C₂)(cycloalkyl),        —(C₀-C₂)(heterocyclo), —(C₀-C₂)(aryl), —(C₀-C₂)(heteroaryl),        —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(C₀-C₂)(cycloalkyl),        —O—(C₀-C₂)(heterocyclo), —O—(C₀-C₂)(aryl), or        —O—(C₀-C₂)(heteroaryl), each of which can be optionally        substituted;    -   R⁴ is a monophosphate, diphosphate, triphosphate, or a        stabilized phosphate prodrug, including but not limited to a        phosphoramidate, a thiophosphoramidate, or any other moiety that        is metabolized to a monophosphate, diphosphate or triphosphate        in vivo in the host human or animal; or    -   R³ and R⁴ together with the oxygens that they are bonded to can        form a 3′,5′-cyclic prodrug, including but not limited to, a        3′,5′-cyclic phosphate prodrug;    -   R¹² is CH₃, CH₂F, CHF₂, CF₃, or ethynyl.

A stabilized phosphate prodrug is any moiety that can deliver a mono,di, or triphosphate.

In another embodiment, compounds of Formula Ia are disclosed:

-   -   wherein:    -   Y, R³ and R⁴ are as defined above.

In another embodiment, compounds of Formula Ib are disclosed:

-   -   wherein:    -   Y, R³ and R⁴ are as defined above.

In another embodiment, the compound is according to Formula Ic:

-   -   wherein:    -   R⁷ is hydrogen, C₁₋₆alkyl; C₃₋₇cycloalkyl; heteroaryl,        heterocyclic, or aryl, which includes, but is not limited to,        phenyl or naphthyl, where phenyl or naphthyl are optionally        substituted with C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl,        C₁₋₆alkoxy, F, Cl, Br, I, nitro, cyano, C₁₋₆haloalkyl,        —N(R^(7′))₂, C₁₋₆acylamino, NHSO₂C₁₋₆alkyl, —SO₂N(R^(7′))₂,        COR^(7″), and —SO₂C₁₋₆alkyl; (R^(7′) is independently hydrogen        or C₁₋₆alkyl; R^(7″) is —OR¹¹ or —N(R⁷)₂);    -   R⁸ is hydrogen, C₁₋₆alkyl, or R^(9a) or R^(9b) and R⁸ together        are (CH₂)_(n) so as to form a cyclic ring that includes the        adjoining N and C atoms; where n is 2 to 4;    -   R^(9a) and R^(9b) are (i) independently selected from hydrogen,        C₁₋₆alkyl, cycloalkyl, —(CH₂)_(c)(NR^(9′))₂, C₁₋₆hydroxyalkyl,        —CH₂SH, —(CH₂)₂S(O)(Me, —(CH₂)₃NHC(═NH)NH₂,        (1H-indol-3-yl)methyl, (1H-imidazol-4-yl)methyl,        —(CH₂)_(c)COR^(9″), aryl and aryl(C₁₋₃alkyl)-, the aryl groups        can be optionally substituted with a group selected from        hydroxyl, C₁₋₆alkyl, C₁₋₆alkoxy, halogen, nitro and cyano; (ii)        R^(9a) and R^(9b) both are C₁₋₆alkyl; (iii) R^(9a) and R^(9b)        together are (CH₂)_(r) so as to form a spiro ring; (iv) R^(9a)        is hydrogen and R^(9b) and R⁸ together are (CH₂)_(n) so as to        form a cyclic ring that includes the adjoining N and C atoms (v)        R^(9b) is hydrogen and R^(9a) and R⁸ together are (CH₂)_(n) so        as to form a cyclic ring that includes the adjoining N and C        atoms, where c is 1 to 6, n is 2 to 4, r is 2 to 5 and where        R^(9′) is independently hydrogen or C₁₋₆ alkyl and R^(9″) is        —OR¹¹ or —N(R^(11′))₂); (vi) R^(9a) is hydrogen and R^(9b) is        hydrogen, CH₃, CH₂CH₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃,        CH₂Ph, CH₂-indol-3-yl, —CH₂CH₂SCH₃, CH₂CO₂H, CH₂C(O)NH₂,        CH₂CH₂COOH, CH₂CH₂C(O)NH₂, CH₂CH₂CH₂CH₂NH₂,        —CH₂CH₂CH₂NHC(NH)NH₂, CH₂-imidazol-4-yl, CH₂OH, CH(OH)CH₃,        CH₂((4′-OH)-Ph), CH₂SH, or lower cycloalkyl; or (vii) R^(9a) is        CH₃, CH₂CH₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂Ph,        CH₂-indol-3-yl, —CH₂CH₂SCH₃, CH₂CO₂H, CH₂C(O)NH₂, CH₂CH₂COOH,        CH₂CH₂C(O)NH₂, CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂NHC(NH)NH₂,        CH₂-imidazol-4-yl, CH₂OH, CH(OH)CH₃, CH₂((4′-OH)-Ph), CH₂SH, or        lower cycloalkyl and R^(9b) is hydrogen;    -   R¹⁰ is hydrogen, C₁₋₆alkyl optionally substituted with an        alkoxy, di(lower alkyl)-amino, or halogen, C₁₋₆haloalkyl,        C₃₋₇cycloalkyl, heterocycloalkyl, aminoacyl, aryl, such as        phenyl, heteroaryl, such as, pyridinyl, substituted aryl, or        substituted heteroaryl;    -   R¹¹ is an optionally substituted C₁₋₆alkyl, an optionally        substituted cycloalkyl; an optionally substituted C₂₋₆alkynyl,        an optionally substituted C₂₋₆alkenyl, or optionally substituted        acyl, which includes but is not limited to C(O)(C₁₋₆ alkyl); and    -   Y, R³ and R¹² are as defined herein.

In one embodiment, compounds of Formula II are disclosed:

-   -   wherein:    -   Y is NR¹R²;    -   R¹ is C₁-C₅alkyl (including methyl, ethyl, n-propyl, iso-propyl,        n-butyl, iso-butyl, sec-butyl, tert-butyl and pentyl),        C₁-C₅haloalkyl (including CH₂F, CHF₂, CF₃, CH₂CF₃, CF₂CH₃ and        CF₂CF₃), C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(heterocycle),        —(C₀-C₂alkyl)(aryl), —(C₀-C₂alkyl)(heteroaryl), —OR²⁵,        —C(O)R^(3C) (including —C(O)CH₃, —C(O)CH₂CH₃—C(O)CH(CH₃)₂,        —C(O)OCH₃, —C(O)OC₂H₅, —C(O)OC₃H₇, —C(O)OC₄H₉, and —C(O)OC₅H₁₁),        —C(S)R^(3D), or —SO₂R²⁸ each of which can be optionally        substituted;    -   R² is hydrogen, optionally substituted C₁-C₅alkyl (including        methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,        sec-butyl, tert-butyl and pentyl), C₁-C₅haloalkyl (including        CHF₂, CHF₂, CF₃, CH₂CF₃ and CF₂CF₃), optionally substituted        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), optionally substituted        —(C₀-C₂alkyl)(heterocycle), optionally substituted        —(C₀-C₂alkyl)(aryl), optionally substituted        —(C₀-C₂alkyl)(heteroaryl), —C(O)R^(3C) (including —C(O)CH₃,        —C(O)CH₂CH₃—C(O)CH(CH₃)₂, —C(O)OCH₃, —C(O)OC₂H₅, —C(O)OC₃H₇,        —C(O)OC₄H₉, and —C(O)OC₅H₁₁), —C(S)R^(3D), or —SO₂R²⁸; and        wherein at least one of R¹ and R² is methyl, CH₂F, CHF₂ or CF₃;    -   R³ is hydrogen,

diphosphate, triphosphate, an optionally substituted carbonyl linkedamino acid, or —C(O)R^(3C);

-   -   R^(3A) can be selected from O⁻, OH, an —O-optionally substituted        aryl, an —O-optionally substituted heteroaryl, or an optionally        substituted heterocyclyl;    -   R^(3B) can be selected from O⁻, OH, an optionally substituted        N-linked amino acid or an optionally substituted N-linked amino        acid ester;    -   R^(3C) is alkyl, alkenyl, alkynyl, —(C₀-C₂)(cycloalkyl),        —(C₀-C₂)(heterocyclo), —(C₀-C₂)(aryl), —(C₀-C₂)(heteroaryl),        —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(C₀-C₂)(cycloalkyl),        —O—(C₀-C₂)(heterocyclo), —O—(C₀-C₂)(aryl),        —O—(C₀-C₂)(heteroaryl), —S-alkyl, —S-alkenyl, —S-alkynyl,        —S—(C₀-C₂)(cycloalkyl), —S—(C₀-C₂)(heterocycle),        —S—(C₀-C₂)(aryl), or —S—(C₀-C₂)(heteroaryl) each of which can be        optionally substituted;    -   R^(3D) is alkyl, alkenyl, alkynyl, —(C₀-C₂)(cycloalkyl),        —(C₀-C₂)(heterocyclo), —(C₀-C₂)(aryl), —(C₀-C₂)(heteroaryl),        —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(C₀-C₂)(cycloalkyl),        —O—(C₀-C₂)(heterocyclo), —O—(C₀-C₂)(aryl), or        —O—(C₀-C₂)(heteroaryl), each of which can be optionally        substituted;    -   R⁴ is a monophosphate, diphosphate, triphosphate, or a        stabilized phosphate prodrug, including but not limited to a        phosphoramidate, a thiophosphoramidate, or any other moiety that        is metabolized to a monophosphate, diphosphate or triphosphate        in vivo in the host human or animal; or    -   R³ and R⁴ together with the oxygens that they are bonded to can        form a 3′,5′-cyclic prodrug, including but not limited to, a        3′,5′-cyclic phosphate prodrug;    -   R⁵ is C₁-C₅alkyl (including methyl, ethyl, n-propyl, iso-propyl,        n-butyl, iso-butyl, sec-butyl, tert-butyl and pentyl),        C₁-C₅haloalkyl (including CHF₂, CHF₂, CF₃, CH₂CF₃ and CF₂CF₃),        C₂-C₆ alkenyl, C₂-C₆ alkynyl, —(C₀-C₂alkyl)(C₃-C₆cycloalkyl),        —(C₀-C₂alkyl)(heterocycle), —(C₀-C₂alkyl)(aryl),        —(C₀-C₂alkyl)(heteroaryl), —OR²⁵, —C(O)R^(3C) (including        —C(O)CH₃, —C(O)CH₂CH₃—C(O)CH(CH₃)₂, —C(O)OCH₃, —C(O)OC₂H₅,        —C(O)OC₃H₇, —C(O)OC₄H₉, and —C(O)OC₅H₁₁), —C(S)R^(3D), or        —SO₂R²⁸ each of which can be optionally substituted;    -   R⁶ is hydrogen, optionally substituted C₁-C₅alkyl (including        methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,        sec-butyl, tert-butyl and pentyl), C₁-C₅haloalkyl (including        CHF₂, CH₂F, CF₃, CH₂CF₃ and CF₂CF₃), optionally substituted        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), optionally substituted        —(C₀-C₂alkyl)(heterocycle), optionally substituted        —(C₀-C₂alkyl)(aryl), optionally substituted        —(C₀-C₂alkyl)(heteroaryl), —C(O)R^(3C) (including —C(O)CH₃,        —C(O)CH₂CH₃—C(O)CH(CH₃)₂, —C(O)OCH₃, —C(O)OC₂H, —C(O)OC₃H₇,        —C(O)OC₄H₉, and —C(O)OC₅H₁₁), —C(S)R^(3D), or —SO₂R²⁸; or    -   R⁵ and R⁶ together with the nitrogen that they are bonded to can        form a heterocyclic ring;    -   R¹² is CH₃, CH₂F, CHF₂, CF₃, or ethynyl;    -   R²² is Cl, Br, F, CN, N₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, —(C₀-C₂alkyl)(C₃-C₆cycloalkyl),        —(C₀-C₂alkyl)(C₃-C₆heterocycle), —(C₀-C₂alkyl)(aryl),        —(C₀-C₂alkyl)(heteroaryl); —ONHC(═O)OR²³, —NHOR²⁴, —OR²⁵, —SR²⁵,        —NH(CH₂)₁₋₄N(R²⁶)₂, —NHNHR²⁶, —N═NR²⁷, —NHC(O)NHNHR²⁷,        —NHC(S)NHNHR²⁷, —C(O)NHNHR²⁷, —NR²⁷SO₂R²⁸, —SO₂NR²⁷R²⁹,        —C(O)NR²⁷R²⁹, —CO₂R²⁹, —SO₂R²⁹,

—P(O)H(OR²⁹), —P(O)(OR²⁹)(OR³⁰), —P(O)(OR²⁹)(NR²⁹R³⁰) or —NR⁵R⁶;

-   -   for example including but not limited to the following        embodiments, chloro, bromo, fluoro, cyano, azido, ethyl,        n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl        and n-pentyl, 1,1-dimethylpropyl, 2,2-dimtheylpropyl,        3-methylbutyl, 1-methylbutyl, 1-ethylpropyl, vinyl, allyl,        1-butynyl, 2-butynyl, acetylenyl, cyclopropyl, cyclobutyl,        cyclopentyl, cyclohexyl, —(CH₂)-cyclopropyl, —(CH₂)-cyclobutyl,        —(CH₂)-cyclopentyl, —(CH₂)-cyclohexyl, aziridine, oxirane,        thiirane, azetidine, oxetane, thietane, pyrrolidine,        tetrahydrofuran, thiolane, pyrazolidine, piperidine, oxane,        thiane, —(CH₂)-aziridine, —(CH₂)-oxirane, —(CH₂)-thiirane,        —(CH₂)-azetidine, —(CH₂)-oxetane, —(CH₂)-thietane,        —(CH₂)-pyrrolidine, —(CH₂)-tetrahydrofuran, —(CH₂)-thiolane,        —(CH₂)-pyrazolidine, —(CH₂)-piperidine, —(CH₂)-oxane,        —(CH₂)-thiane, phenyl, pyridyl, —ONHC(═O)OCH₃, —ONHC(═O)OCH₂CH₃,        —NHOH, NHOCH₃, —OCH₃, OC₂H₅, —OPh, OCH₂Ph, —SCH₃, —SC₂H₅, —SPh,        SCH₂Ph, —NH(CH₂)₂NH₂, —NH(CH₂)₂N(CH₃)₂, —NHNH₂, —NHNHCH₃, —N═NH,        —N═NCH₃, —N═NCH₂CH₃, —NHC(O)NHNH₂, —NHC(S)NHNH₂, —C(O)NHNH₂,        —NHSO₂CH₃, —NHSO₂CH₂CH₃, —SO₂NHCH₃, —SO₂N(CH₃)₂, —C(O)NH₂,        —C(O)NHCH₃, —C(O)N(CH₃)₂, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂Ph,        —CO₂CH₂Ph, —SO₂CH₃, —SO₂CH₂CH₃, —SO₂Ph, —SO₂CH₂Ph,

—P(O)H(OH), —P(O)H(OCH₃), —P(O)(OH)(OH), —P(O)(OH)(OCH₃),—P(O)(OCH₃)(OCH₃), —P(O)(OH)(NH₂), —P(O)(OH)(NHCH₃), —P(O)(OH)N(CH₃)₂,—NHC(O)CH₃, —NHC(O)CH₂CH₃, —NHC(O)CH(CH₃)₂, —NHC(O)OCH₃, —NHC(O)OCH₂CH₃,—NHC(O)OCH(CH₃)₂, —NHC(O)OCH₂CH₂CH₃, —NHC(O)OCH₂CH₂CH₂CH₃ and—NHC(O)OCH₂CH₂CH₂CH₂CH₃;

-   -   R²³ is C₁-C₅alkyl, —(C₀-C₂alkyl)(C₃-C₆cycloalkyl),        —(C₀-C₂alkyl)(heterocycle)-(C₀₋₂alkyl)(aryl) or        —(C₀-C₂alkyl)(heteroaryl) each of which can be optionally        substituted;    -   R²⁴ is hydrogen, C₁-C₆ alkyl, —(C₀-C₂alkyl)(C₃-C₆cycloalkyl),        —(C₁-C₂alkyl)(C₃-C₆heterocycle) —(C₀-C₂alkyl)(aryl) or        —(C₀-C₂alkyl)(heteroaryl) wherein except for the hydrogen each        of which can be optionally substituted;    -   R²⁵ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(C₃-C₆heterocycle),        —(C₀-C₂alkyl)(aryl) or —(C₀-C₂alkyl)(heteroaryl) wherein except        for the hydrogen each of which can be optionally substituted;    -   R²⁶ is independently selected from hydrogen, C₁-C₆alkyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(heterocycle),        —(C₀-C₂alkyl)(aryl), or —(C₀-C₂alkyl)(heteroaryl) wherein except        for the hydrogen each of which can be optionally substituted;    -   R²⁷ hydrogen or optionally substituted C₁-C₆ alkyl;    -   R²⁸ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(C₃-C₆heterocycle),        —(C₀-C₂alkyl)(aryl) or —(C₀-C₂alkyl)(heteroaryl) each of which        can be optionally substituted;    -   R²⁹ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃-C₆cycloalkyl), —(C₀-C₂alkyl)(C₃-C₆heterocycle),        —(C₀-C₂alkyl)(aryl) or —(C₀-C₂alkyl)(heteroaryl) wherein except        for the hydrogen each of which can be optionally substituted; or    -   R²⁷ and R²⁹ together with the nitrogen that they are bonded to        can form a heterocyclic ring;    -   R³⁰ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        —(C₀-C₂alkyl)(C₃C₆cycloalkyl), —(C₀-C₂alkyl)(C₃-C₆heterocycle),        —(C₀-C₂alkyl)(aryl) or —(C₀-C₂alkyl)(heteroaryl) wherein except        for the hydrogen each of which can be optionally substituted; or    -   R²⁹ and R³⁰ can be bonded together to form a heterocyclic ring;    -   x is 1, 2 or 3.

In another embodiment, compounds of Formula IIa are disclosed:

-   -   wherein:    -   Y, R³, R⁴ and R²² are as defined above.

In another embodiment, compounds of Formula IIb are disclosed:

-   -   wherein:    -   Y, R³, R⁴ and R²² are as defined above.

In a typical embodiment, the compound is a β-D isomer with reference tothe corresponding nucleoside (i.e., in the naturally occurringconfiguration). In an alternative configuration, the compound isprovided as a β-L isomer. The compound is typically at least 90% free ofthe opposite enantiomer, and can be at least 98%, 99% or even 100% freeof the opposite enantiomer. Unless described otherwise, the compound isat least 90% free of the opposite enantiomer.

In another embodiment, the compound is according to Formula III:

-   -   wherein:    -   R⁷ is hydrogen, C₁₋₆alkyl; C₃₋₇cycloalkyl; heteroaryl,        heterocyclic, or aryl, which includes, but is not limited to,        phenyl or naphthyl, where phenyl or naphthyl are optionally        substituted with C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl,        C₁₋₆alkoxy, F, Cl, Br, I, nitro, cyano, C₁₋₆haloalkyl,        —N(R^(7′))₂, C₁₋₆acylamino, NHSO₂C₁₋₆alkyl, —SO₂N(R^(7′))₂,        COR^(7″), and —S₂C₁₋₆alkyl; (R^(7′) is independently hydrogen or        C₁₋₆alkyl; R^(7″) is —OR¹¹ or —N(R⁷)₂);    -   R⁸ is hydrogen, C₁₋₆alkyl, or R^(9a) or R^(9b) and R⁸ together        are (CH₂)_(n) so as to form a cyclic ring that includes the        adjoining N and C atoms; where n is 2 to 4;    -   R^(9a) and R^(9b) are (i) independently selected from hydrogen,        C₁₋₆alkyl, cycloalkyl, —(CH₂)_(c)(NR^(9′))₂, C₁₋₆hydroxyalkyl,        —CH₂SH, —(CH₂)₂S(O)(Me, —(CH₂)₃NHC(═NH)NH₂,        (1H-indol-3-yl)methyl, (1H-imidazol-4-yl)methyl,        —(CH₂)_(c)COR^(9″), aryl and aryl(C₁₋₃alkyl)-, the aryl groups        can be optionally substituted with a group selected from        hydroxyl, C₁₋₆alkyl, C₁₋₆alkoxy, halogen, nitro and cyano; (ii)        R^(9a) and R^(9b) both are C₁₋₆alkyl; (iii) R^(9a) and R^(9b)        together are (CH₂)_(r) so as to form a spiro ring; (iv) R^(9a)        is hydrogen and R^(9b) and R⁸ together are (CH₂)_(n) so as to        form a cyclic ring that includes the adjoining N and C atoms (v)        R^(9b) is hydrogen and R^(9a) and R⁸ together are (CH₂)_(n) so        as to form a cyclic ring that includes the adjoining N and C        atoms, where c is 1 to 6, n is 2 to 4, r is 2 to 5 and where        R^(9′) is independently hydrogen or C₁₋₆ alkyl and R^(9″) is        —OR¹¹ or —N(R^(11′))₂); (vi) R^(9a) is hydrogen and R^(9b) is        hydrogen, CH₃, CH₂CH₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃,        CH₂Ph, CH₂-indol-3-yl, —CH₂CH₂SCH₃, CH₂CO₂H, CH₂C(O)NH₂,        CH₂CH₂COOH, CH₂CH₂C(O)NH₂, CH₂CH₂CH₂CH₂NH₂,        —CH₂CH₂CH₂NHC(NH)NH₂, CH₂-imidazol-4-yl, CH₂OH, CH(OH)CH₃,        CH₂((4′-OH)-Ph), CH₂SH, or lower cycloalkyl; or (vii) R^(9a) is        CH₃, CH₂CH₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂Ph,        CH₂-indol-3-yl, —CH₂CH₂SCH₃, CH₂CO₂H, CH₂C(O)NH₂, CH₂CH₂COOH,        CH₂CH₂C(O)NH₂, CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂NHC(NH)NH₂,        CH₂-imidazol-4-yl, CH₂OH, CH(OH)CH₃, CH₂((4′-OH)-Ph), CH₂SH, or        lower cycloalkyl and R^(9b) is hydrogen;    -   R¹⁰ is hydrogen, C₁₋₆alkyl optionally substituted with an        alkoxy, di(lower alkyl)-amino, or halogen, C₁₋₆haloalkyl,        C₃₋₇cycloalkyl, heterocycloalkyl, aminoacyl, aryl, such as        phenyl, heteroaryl, such as, pyridinyl, substituted aryl, or        substituted heteroaryl;    -   R¹¹ is an optionally substituted C₁₋₆alkyl, an optionally        substituted cycloalkyl; an optionally substituted C₂₋₆alkynyl,        an optionally substituted C₂₋₆alkenyl, or optionally substituted        acyl, which includes but is not limited to C(O)(C₁₋₆ alkyl); and    -   Y, R³ R¹² and R²² are as defined above.

In one embodiment, compounds of Formula IV are disclosed:

-   -   wherein the variables Y, R³, R⁷, R¹, R^(9a), R^(9b), R¹⁰ and R²²        are described herein.

In one embodiment, compounds of Formula V are disclosed:

-   -   wherein the variables Y, R³, R⁷, R¹, R^(9a), R^(9b), R¹⁰ and R²²        are described herein.

In an alternative embodiment, compounds, methods, and compositions areprovided for the treatment of a host infected with or exposed tohepatitis C.

In one embodiment, compounds of Formula VI are disclosed:

-   -   wherein:    -   R⁴¹ is halogen (in particular F or Cl), OR³ (including OH), N₃,        NH₂ or CN; and    -   the variables Y, R³, R⁴, and R¹² are described herein.

In one embodiment, compounds of Formula VII are disclosed:

Wherein the variables Y, R³, R⁴, R¹² and R⁴¹ are described herein.

Metabolism ofβ-D-2′-deoxy-2′-α-fluoro-2′-β-C-substituted-N⁶-substituted-2,6-diaminopurinenucleotides

The metabolism of theβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diaminopurinenucleoside phosphoramidate involves the production of a 5′-monophosphateand the subsequent anabolism of the N⁶-methyl-2,6-diaminopurine base togenerate the β-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-guanine nucleoside asthe 5′-monophosphate. The monophosphate is then further anabolized tothe active species; the 5′-triphosphate. Theβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-guanine triphosphate has anIC₅₀=0.15 μM against the HCV genotype 1b NS5B polymerase. The metabolicpathway for theβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diaminopurinenucleoside phosphoramidate is illustrated in Scheme 1 below.

The metabolism of theβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-dimethyl-2,6-diaminopurinenucleotide involves both the formation of theβ-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-dimethyl-2,6-diaminopurinenucleoside triphosphate as well as the generation of the correspondingguanine nucleoside triphosphate. These metabolic pathways areillustrated in Schemes 2 and 3 below.

Stabilized Phosphate Prodrugs

Stabilized phosphate prodrugs are moieties that can deliver a mono, di,or triphosphate in vivo. For example, McGuigan has disclosedphosphoramidates in U.S. Pat. Nos. 8,933,053; 8,759,318; 8,658,616;8,263,575; 8,119,779; 7,951,787 and 7,115,590. Alios has disclosedthiophosphoramidates in U.S. Pat. Nos. 8,895,723 and 8,871,737incorporated by reference herein. Alios has also disclosed cyclicnucleotides in U.S. Pat. No. 8,772,474 incorporated by reference herein.Idenix has disclosed cyclic phosphoramidates and phosphoramidate/SATEderivatives in WO 2013/177219 incorporated by reference herein. Idenixhas also disclosed substituted carbonyloxymethylphosphoramidatecompounds in WO 2013/039920 incorporated by reference herein. Hostetlerhas disclosed lipid phosphate prodrugs, see, for example, U.S. Pat. No.7,517,858. Hostetler has also disclosed lipid conjugates of phosphonateprodrugs, see, for example, U.S. Pat. Nos. 8,889,658; 8,846,643;8,710,030; 8,309,565; 8,008,308; and 7,790,703. Emory University hasdisclosed nucleotide sphingoid and lipid derivatives in WO 2014/124430.RFS Pharma has disclosed purine nucleoside monophosphate prodrugs in WO2010/091386. Cocrystal Pharma Inc. has also disclosed purine nucleosidemonophosphate prodrugs in U.S. Pat. No. 9,173,893 incorporated byreference herein. HepDirect™ technology is disclosed in the article“Design, Synthesis, and Characterization of a Series of CytochromeP(450) 3A-Activated Prodrugs (HepDirect Prodrugs) Useful for TargetingPhosph(on)ate-Based Drugs to the Liver,” (J. Am. Chem. Soc. 126,5154-5163 (2004). Additional phosphate prodrugs include, but are notlimited to phosphate esters, 3′,5′-cyclic phosphates including CycloSAL,SATE derivatives (S-acyl-2thioesters) and DTE (dithiodiethyl) prodrugs.For literature reviews that disclose non-limiting examples see: A. Rayand K. Hostetler, “Application of kinase bypass strategies to nucleosideantivirals,” Antiviral Research (2011) 277-291; M. Sofia, “Nucleotideprodrugs for HCV therapy,” Antiviral Chemistry and Chemotherapy 2011;22-23-49; and S. Peyrottes et al., “SATE Pronucleotide Approaches: AnOverview,” Mini Reviews in Medicinal Chemistry 2004, 4, 395.

In one embodiment, a 5′-prodrug described in any of these patent filingsor literature can be used in the R⁴ position of the presented compounds.

In one alternative embodiment, the stabilized phosphate prodrugs,include, but are not limited to those described in U.S. Pat. Nos.9,173,893 and 8,609,627, incorporated by reference herein, including forprocesses of preparation. For example, 5′-prodrugs of Formula I-V can berepresented by the group:

In an alternate embodiment, 3′,5′-prodrugs of Formula I-V can berepresented by the group:

wherein:when chirality exists at the phosphorous center it may be wholly orpartially R_(p) or S_(p) or any mixture thereof.

-   -   Z is O or S;    -   R³³ is selected from OR³⁴,

fatty alcohol derived (for example but not limited to:

wherein R³⁴, R³⁵, and R³⁶ are as defined below;R³¹ and R³², when administered in vivo, are capable of providing thenucleoside monophosphate or thiomonophosphate, which may or may not bepartially or fully resistant to 6-NH₂ deamination in a biologicalsystem. Representative R³¹ and R³² are independently selected from: (a)OR³⁴ where R³⁴ is selected from H, Li, Na, K, phenyl and pyridinyl;phenyl and pyridinyl are substituted with one to three substituentsindependently selected from the group consisting of (CH₂)₀₋₆CO₂R³⁷ and(CH₂)₀₋₆CON(R³⁷)₂;R³⁷ is independently H, C-2 alkyl, the carbon chain derived from a fattyalcohol (such as oleyl alcohol, octacosanol, triacontanol, linoleylalcohol, and etc) or C₁₋₂₀ alkyl substituted with a lower alkyl, alkoxy,di(lower alkyl)-amino, fluoro, C₃₋₁₀ cycloalkyl, cycloalkyl alkyl,cycloheteroalkyl, aryl, such as phenyl, heteroaryl, such as, pyridinyl,substituted aryl, or substituted heteroaryl; wherein the substituentsare C₁₋₅ alkyl, or C₁₋₅ alkyl substituted with a lower alkyl, alkoxy,di(lower alkyl)-amino, fluoro, C₃₋₁₀ cycloalkyl, or cycloalkyl;(b)

or(c) the ester of a D-amino acid or L-amino acid

where R³⁶ is restricted to those sidechains occurring in natural L-aminoacids, andR³⁵ is H, C₁₋₂₀ alkyl, the carbon chain derived from a fatty alcohol(such as oleyl alcohol, octacosanol, triacontanol, linoleyl alcohol, andetc) or C₁₋₂₀ alkyl substituted with a lower alkyl, alkoxy, di(loweralkyl)-amino, fluoro, C₃₋₁₀ cycloalkyl, cycloalkyl alkyl,cycloheteroalkyl, aryl, such as phenyl, heteroaryl, such as, pyridinyl,substituted aryl, or substituted heteroaryl; wherein the substituentsare C₁₋₅ alkyl, or C₁₋₅ alkyl substituted with a lower alkyl, alkoxy,di(lower alkyl)-amino, fluoro, C₃₋₁₀ cycloalkyl, or cycloalkyl;(d) R³¹ and R³² can come together to form a ring

where R³⁸ is H, C₁₋₂₀ alkyl, C₁₋₂₀ alkenyl, the carbon chain derivedfrom a fatty alcohol (such as oleyl alcohol, octacosanol, triacontanol,linoleyl alcohol, etc) or C₁₋₂₀ alkyl substituted with a lower alkyl,alkoxy, di(lower alkyl)-amino, fluoro, C₃₋₁₀ cycloalkyl, cycloalkylalkyl, cycloheteroalkyl, aryl, such as phenyl, heteroaryl, such as,pyridinyl, substituted aryl, or substituted heteroaryl; wherein thesubstituents are C₁₋₅ alkyl, or C₁₋₅ alkyl substituted with a loweralkyl, alkoxy, di(lower alkyl)-amino, fluoro, C₃₋₁₀ cycloalkyl, orcycloalkyl;(e) R³¹ and R³² can come together to form a ring selected from

where R³⁹ is O or NH andR⁴⁰ is selected from H, C₁₋₂₀ alkyl, C₁₋₂₀ alkenyl, the carbon chainderived from a fatty acid (such as oleic acid, linoleic acid, and thelike), and C₁₋₂₀ alkyl substituted with a lower alkyl, alkoxy, di(loweralkyl)-amino, fluoro, C₃₋₁₀ cycloalkyl, cycloalkyl alkyl,cycloheteroalkyl, aryl, such as phenyl, heteroaryl, such as pyridinyl,substituted aryl, or substituted heteroaryl; wherein the substituentsare C₁₋₅ alkyl, or C₁₋₅ alkyl substituted with a lower alkyl, alkoxy,di(lower alkyl)-amino, fluoro, C₃₋₁₀ cycloalkyl, or cycloalkyl.

The compounds can be prepared, for example, by preparing the 5′-OHanalogs, then converting these to the monophosphate analogs.

Embodiments

In particular embodiments:

-   -   (i) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is hydrogen, R³        is hydrogen, R⁴ is a stabilized phosphate prodrug;    -   (ii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is hydrogen, R³        is hydrogen, and R⁴ is a stabilized thiophosphate prodrug;    -   (iii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is hydrogen,        R³ is hydrogen, and R⁴ is a phosphoramidate;    -   (iv) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is hydrogen, R³        is hydrogen, and R⁴ is a thiophosphoramidate:    -   (v) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is hydrogen, R³        is hydrogen, and R⁴ is a monophosphate;    -   (vi) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is hydrogen, R³        is hydrogen, and R⁴ is a diphosphate;    -   (vii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is hydrogen,        R³ is hydrogen, and R⁴ is a triphosphate;    -   (viii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is methyl, R³        is hydrogen, R⁴ is a stabilized phosphate prodrug;    -   (ix) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is methyl, R³        is hydrogen, and R⁴ is a stabilized thiophosphate prodrug;    -   (x) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is methyl, R³ is        hydrogen, and R⁴ is a phosphoramidate;    -   (xi) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is methyl, R³        is hydrogen, and R⁴ is a thiophosphoramidate:    -   (xii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is methyl, R³        is hydrogen, and R⁴ is a monophosphate;    -   (xiii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is methyl, R³        is hydrogen, and R⁴ is a diphosphate;    -   (xiv) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is methyl, R³        is hydrogen, and R⁴ is a triphosphate;    -   (xv) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is cyclopropyl,        R³ is hydrogen, R⁴ is a stabilized phosphate prodrug;    -   (xvi) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is        cyclopropyl, R³ is hydrogen, and R⁴ is a stabilized        thiophosphate prodrug;    -   (xvii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is        cyclopropyl, R³ is hydrogen, and R⁴ is a phosphoramidate;    -   (xviii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is        cyclopropyl, R³ is hydrogen, and R⁴ is a thiophosphoramidate:    -   (xix) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is        cyclopropyl, R³ is hydrogen, and R⁴ is a monophosphate;    -   (xx) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is cyclopropyl,        R³ is methyl, and R⁴ is a diphosphate;    -   (xxi) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is        cyclopropyl, R³ is hydrogen, and R⁴ is a triphosphate;    -   (xxii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is propyl, R³        is hydrogen, R⁴ is a stabilized phosphate prodrug;    -   (xxiii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is propyl,        R³ is hydrogen, and R⁴ is a stabilized thiophosphate prodrug;    -   (xxiv) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is propyl, R³        is hydrogen, and R⁴ is a phosphoramidate;    -   (xxv) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is propyl, R³        is hydrogen, and R⁴ is a thiophosphoramidate:    -   (xxvi) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is propyl, R³        is hydrogen, and R⁴ is a monophosphate;    -   (xxvii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is propyl,        R³ is hydrogen, and R⁴ is a diphosphate;    -   (xxviii) in Formula Ia, Y is NR¹R², Y is NR¹R², R¹ is methyl, R²        is propyl, R³ is hydrogen, and R⁴ is a triphosphate;    -   (xxix) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is ethyl, R³        is hydrogen, R⁴ is a stabilized phosphate prodrug;    -   (xxx) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is ethyl, R³        is hydrogen, and R⁴ is a stabilized thiophosphate prodrug;    -   (xxxi) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is ethyl, R³        is hydrogen, and R⁴ is a phosphoramidate;    -   (xxxii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is ethyl, R³        is hydrogen, and R⁴ is a thiophosphoramidate:    -   (xxxiii) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is ethyl,        R³ is hydrogen, and R⁴ is a monophosphate;    -   (xxxiv) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is ethyl, R³        is hydrogen, and R⁴ is a diphosphate;    -   (xxxv) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is ethyl, R³        is hydrogen, and R⁴ is a triphosphate;    -   (xxxvi) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is methyl,        R³ is hydrogen, R⁴ is a stabilized phosphate prodrug;    -   (xxxvii) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is methyl,        R³ is hydrogen, and R⁴ is a stabilized thiophosphate prodrug;    -   (xxxviii) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is methyl,        R³ is hydrogen, and R⁴ is a phosphoramidate;    -   (xxxix) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is methyl,        R³ is hydrogen, and R⁴ is a thiophosphoramidate:    -   (xl) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is methyl, R³        is hydrogen, and R⁴ is a monophosphate;    -   (xli) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is methyl, R³        is hydrogen, and R⁴ is a diphosphate;    -   (xlii) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is methyl, R³        is hydrogen, and R⁴ is a triphosphate;    -   (xliii) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is hydrogen,        R³ is hydrogen, R⁴ is a stabilized phosphate prodrug;    -   (xliv) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is hydrogen,        R³ is hydrogen, and R⁴ is a stabilized thiophosphate prodrug;    -   (xlv) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is hydrogen,        R³ is hydrogen, and R⁴ is a phosphoramidate;    -   (xlvi) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is hydrogen,        R³ is hydrogen, and R⁴ is a thiophosphoramidate:    -   (xlvii) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is hydrogen,        R³ is hydrogen, and R⁴ is a monophosphate;    -   (xlviii) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is        hydrogen, R³ is hydrogen, and R⁴ is a diphosphate;    -   (xlix) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is hydrogen,        R³ is hydrogen, and R⁴ is a triphosphate;    -   (1) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is cyclopropyl,        R³ is hydrogen, R⁴ is a stabilized phosphate prodrug;    -   (li) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is cyclopropyl,        R³ is hydrogen, and R⁴ is a stabilized thiophosphate prodrug;    -   (lii) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is        cyclopropyl, R³ is hydrogen, and R⁴ is a phosphoramidate;    -   (liii) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is        cyclopropyl, R³ is hydrogen, and R⁴ is a thiophosphoramidate:    -   (liv) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is        cyclopropyl, R³ is hydrogen, and R⁴ is a monophosphate;    -   (lv) in Formula Ib, Y is NR¹R², R¹ is methyl, R² is cyclopropyl,        R³ is methyl, and R⁴ is a diphosphate;    -   (lvi) in Formula Ia, Y is NR¹R², R¹ is methyl, R² is        cyclopropyl, R³ is hydrogen, and R⁴ is a triphosphate.

In alternative embodiments of any of the above, the compound has an R²²substituent. In some of these specific embodiments, the R²² is F, amideor carbamate. In other specific aspects of the embodiments above, R²² ischloro, bromo, cyano, azido, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, tert-butyl and n-pentyl, 1,1-dimethylpropyl,2,2-dimtheylpropyl, 3-methylbutyl, 1-methylbutyl, 1-ethylpropyl, vinyl,allyl, 1-butynyl, 2-butynyl, acetylenyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, —(CH₂)-cyclopropyl, —(CH₂)-cyclobutyl,—(CH₂)-cyclopentyl, —(CH₂)-cyclohexyl, aziridine, oxirane, thiirane,azetidine, oxetane, thietane, pyrrolidine, tetrahydrofuran, thiolane,pyrazolidine, piperidine, oxane, thiane, —(CH₂)-aziridine,—(CH₂)-oxirane, —(CH₂)-thiirane, —(CH₂)-azetidine, —(CH₂)-oxetane,—(CH₂)-thietane, —(CH₂)-pyrrolidine, —(CH₂)-tetrahydrofuran,—(CH₂)-thiolane, —(CH₂)-pyrazolidine, —(CH₂)-piperidine, —(CH₂)-oxane,—(CH₂)-thiane, phenyl, pyridyl, —ONHC(═O)OCH₃, —ONHC(═O)OCH₂CH₃, —NHOH,NHOCH₃, —OCH₃, OC₂H₅, —OPh, OCH₂Ph, —SCH₃, —SC₂H₅, —SPh, SCH₂Ph,—NH(CH₂)₂NH₂, —NH(CH₂)₂N(CH₃)₂, —NHNH₂, —NHNHCH₃, —N═NH, —N═NCH₃,—N═NCH₂CH₃, —NHC(O)NHNH₂, —NHC(S)NHNH₂, —C(O)NHNH₂, —NHSO₂CH₃,—NHSO₂CH₂CH₃, —SO₂NHCH₃, —SO₂N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂Ph, CO₂CH₂Ph, —SO₂CH₃,—SO₂CH₂CH₃, —SO₂Ph, —SO₂CH₂Ph,

—P(O)H(OH), —P(O)H(OCH₃), —P(O)(OH)(OH), —P(O)(OH)(OCH₃),—P(O)(OCH₃)(OCH₃), —P(O)(OH)(NH₂), —P(O)(OH)(NHCH₃), —P(O)(OH)N(CH₃)₂,—NHC(O)CH₃, —NHC(O)CH₂CH₃, —NHC(O)CH(CH₃)₂, —NHC(O)OCH₃, —NHC(O)OCH₂CH₃,—NHC(O)OCH(CH₃)₂, —NHC(O)OCH₂CH₂CH₃, —NHC(O)OCH₂CH₂CH₂CH₃ and—NHC(O)OCH₂CH₂CH₂CH₂CH₃;

In alternative embodiments of compounds (i) through (lvi), anL-nucleoside is used in Formula I-VII.

In an alternate embodiment, the Formula I R¹² variable is CH₂F.

In an alternate embodiment, the Formula I R¹² variable is CHF₂.

In an alternate embodiment, the Formula I R¹² variable is CF₃.

In one embodiment, a compound of Formula Ia is provided. Non-limitingexamples of compounds of Formula Ia include:

In one embodiment, a thiophosphoramidate of Formula Ia is provided.Non-limiting examples of thiophosphoramidates of Formula Ia include, butare not limited to:

In one embodiment, a stabilized phosphate prodrug of Formula Ia isprovided. Non-limiting examples of stabilized phosphate prodrugs ofFormula Ia are illustrated below:

In another embodiment, a compound of Formula Ia is provided.Non-limiting examples of compounds of Formula Ia include:

In one embodiment, a thiophosphoramidate of Formula Ia is provided.Non-limiting examples of thiophosphoramidates of Formula Ia include, butare not limited to:

In one embodiment, a stabilized phosphate prodrug of Formula Ia isprovided. Non-limiting examples of stabilized phosphate prodrugs ofFormula Ia are illustrated below:

In one embodiment, a compound of Formula II is provided. Non-limitingexamples of compounds of Formula II include:

In one embodiment, a compound of Formula I is provided. Non-limitingexamples of compounds of Formula I include:

In one embodiment, a compound of Formula II is provided. Non-limitingexamples of compounds of Formula II include.

In one embodiment, and R⁴ is

In one embodiment, a compound of Formula II is provided. Non-limitingexamples of compounds of Formula II include:

In some embodiments, R³ is H and R⁴ is

In some embodiments, R³ is H and R⁴ is

In some embodiments, R³ is H and R⁴ is

In one embodiment, a compound of Formula II is provided. Non-limitingexamples of compounds of Formula II include:

In some embodiments, R³ is H and R⁴ is

In some embodiments, R³ is H and R⁴ is

In some embodiments, R³ is H and R⁴ is

In some embodiments, R¹ is CH₃, R² is H, R³ is H and R⁴ is

In some embodiments, R¹ is CH₃, R² is H, R³ is H and R⁴ is

In some embodiments, R¹ is CH₃, R² is H, R³ is H and R⁴ is

In some embodiments, R¹ is CH₃, R² is CH₃, R³ is H and R⁴ is

In some embodiments, R¹ is CH₃, R² is CH₃, R³ is H and R⁴ is

In some embodiments, R¹ is CH₃, R² is CH₃, R³ is H and R⁴ is

In some embodiments, R¹ is cyclopropyl, R² is CH₃, R³ is H and R⁴ is

In some embodiments, R¹ is cyclopropyl, R² is CH₃, R³ is H and R⁴ is

In some embodiments, R¹ is cyclopropyl, R² is CH₃, R³ is H and R⁴ is

II. Definitions

The following terms are used to describe the present invention. Ininstances where a term is not specifically defined herein, that term isgiven an art-recognized meaning by those of ordinary skill applying thatterm in context to its use in describing the present invention.

The term “alkyl” shall mean within its context, a linear, orbranch-chained fully saturated hydrocarbon radical or alkyl group whichcan be optionally substituted (for example, with halogen, including F).For example, an alkyl group can have 1, 2, 3, 4, 5, 6, 7 or 8 carbonatoms (i.e., C₁-C₈ alkyl), 1, 2, 3, 4, 5 or 6 carbon atoms (i.e., C₁-C₆alkyl) or 1 to 4 carbon atoms (i.e., C₁-C₄ alkyl). Examples of suitablealkyl groups include, but are not limited to, methyl, ethyl, n-propyl,iso-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl,tert-pentyl, neopentyl, hexyl, 2-methylpentyl, 3-methylpentyl,2,2-dimethylbutyl and 2,3-dimethylbutyl.

The term “alkenyl” refers to a non-aromatic hydrocarbon group whichcontains at least one double bond between adjacent carbon atoms and asimilar structure to an alkyl group as otherwise described herein. Forexample, an alkenyl group can have 2 to 8 carbon atoms (i.e., C₂-C₈alkenyl), or 2 to 4 carbon atoms (i.e., C₂-C₄ alkenyl). Examples ofsuitable alkenyl groups include, but are not limited to, ethenyl orvinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), 1-butenyl (—C═CH—CH₂CH₃) and2-butenyl (—CH₂CH═CHCH₂). The alkenyl group can be optionallysubstituted as described herein.

The term “alkynyl” refers to a non-aromatic hydrocarbon group containingat least one triple bond between adjacent carbon atoms and a similarstructure to an alkyl group as otherwise described herein. For example,an alkynyl group can have 2 to 8 carbon atoms (i.e., C₂-C₈ alkyne), or 2to 4 carbon atoms (i.e., C₂-C₄ alkynyl). Examples of alkynyl groupsinclude, but are not limited to, acetylenic or ethynyl and propargyl.The alkynyl group can be optionally substituted as described herein.

The term “acyl” refers to the moiety —C(O)R in which the carbonyl moietyis bonded to R, for example, —C(O)alkyl. R can be selected from alkoxy,alkyl, cycloalkyl, lower alkyl (i.e., C₁-C₄); alkoxyalkyl, includingmethoxymethyl; aralkyl- including benzyl, aryloxyalkyl- such asphenoxymethyl; aryl including phenyl optionally substituted withhalogen, C₁ to C₄ alkyl or C₁ to C₄ alkoxy. In one embodiment, the term“acyl” refers to a mono, di or triphosphate.

The term “lower acyl” refers to an acyl group in which the carbonylmoiety is lower alkyl (i.e., C₁-C₄).

The term “alkoxy” refers to the group —OR′ where —OR′ is —O-alkyl,—O-alkenyl, —O— alkynyl, —O—(C₀-C₂)(cycloalkyl),—O—(C₀-C₂)(heterocyclo), —O—(C₀-C₂)(aryl), or —O—(C₀-C₂)(heteroaryl),each of which can be optionally substituted.

The term “amino” refers to the group —NH₂.

The term “amino acid” or “amino acid residue” refers to a D- orL-natural or non-naturally occurring amino acid. Representative aminoacids include, but are not limited to, alanine, β-alanine, arginine,asparagine, aspartic acid, cysteine, cysteine, glutamic acid, glutamine,glycine, phenylalanine, histidine, isoleucine, lysine, leucine,methionine, proline, serine, threonine, valine, tryptophan, or tyrosine,among others.

The term “azido” refers to the group —N₃.

The term “aryl” or “aromatic”, in context, refers to a substituted (asotherwise described herein) or unsubstituted monovalent aromatic radicalhaving a single ring (e.g., phenyl or benzyl) or condensed rings (e.g.,naphthyl, anthracenyl, phenanthrenyl, etc.) and can be bound to thecompound according to the present invention at any available stableposition on the ring(s) or as otherwise indicated in the chemicalstructure presented. The aryl group can be optionally substituted asdescribed herein.

“Cycloalkyl”, “carbocycle”, or “carbocyclyl” refers to a saturated(i.e., cycloalkyl) or partially unsaturated (e.g., cycloakenyl,cycloalkadienyl, etc.) ring having 3 to 7 carbon atoms as a monocycle.Monocyclic carbocycles have 3 to 7 ring atoms, still more typically 5 or6 ring atoms. Non-limiting examples of cycloalkyl groups includecyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,1-cyclohex-2-enyl, and 1-cyclo-hex-3-enyl.

The term “cyano” refers to the group —CN.

The term “halogen” or “halo” refers to chloro, bromo, fluoro or iodo.

A heteroaryl ring system is a saturated or unsaturated ring with one ormore nitrogen, oxygen, or sulfur atoms in the ring (monocyclic)including but not limited to imidazole, furyl, pyrrole, furanyl, thiene,thiazole, pyridine, pyrimidine, purine, pyrazine, triazole, oxazole, orfused ring systems such as indole, quinoline, etc., among others, whichmay be optionally substituted as described above. Heteroaryl groupsinclude nitrogen-containing heteroaryl groups such as pyrrole, pyridine,pyridone, pyridazine, pyrimidine, pyrazine, pyrazole, imidazole,triazole, triazine, tetrazole, indole, isoindole, indolizine, purine,indazole, quinoline, isoquinoline, quinolizine, phthalazine,naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine,imidazopyridine, imidazotriazine, pyrazinopyridazine, acridine,phenanthridine, carbazole, carbazoline, perimidine, phenanthroline,phenacene, oxadiazole, benzimidazole, pyrrolopyridine, pyrrolopyrimidineand pyridopyrimidine; sulfur-containing aromatic heterocycles such asthiophene and benzothiophene; oxygen-containing aromatic heterocyclessuch as furan, pyran, cyclopentapyran, benzofuran and isobenzofuran; andaromatic heterocycles comprising two or more hetero atoms selected fromamong nitrogen, sulfur and oxygen, such as thiazole, thiadizole,isothiazole, benzoxazole, benzothiazole, benzothiadiazole,phenothiazine, isoxazole, furazan, phenoxazine, pyrazoloxazole,imidazothiazole, thienofuran, furopyrrole, pyridoxazine, furopyridine,furopyrimidine, thienopyrimidine and oxazole, among others, all of whichmay be optionally substituted.

The term “heterocycle” or “heterocyclo” refers to a cyclic group whichcontains at least one heteroatom, i.e., O, N, or S, and may be aromatic(heteroaryl) or non-aromatic. Exemplary non-aromatic heterocyclic groupsfor use in the present invention include, for example, pyrrolidinyl,piperidinyl, piperazinyl, N-methylpiperazinyl, imidazolinyl,pyrazolidinyl, imidazolidinyl, morpholinyl, tetrahydropyranyl,azetidinyl, oxetanyl, oxathiolanyl, pyridone, 2-pyrrolidone,ethyleneurea, 1,3-dioxolane, 1,3-dioxane, 1,4-dioxane, phthalimide, andsuccinimide, among others, all of which may be optionally substituted.

The term “hydroxyl” refers to the group —OH.

The term “nitro” refers to the group —NO₂.

The term “pharmaceutically acceptable salt” or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphoramidate, thiophosphoramidate, phosphateester, salt of an ester, or a related group) of aβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotide which, upon administration to a patient, provides the desiredactive compound. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids, which form aphysiological acceptable anion, for example, tosylate, methanesulfonate,acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including sulfate, nitrate, bicarbonate, and carbonatesalts. Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium, or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

“Pharmaceutically acceptable prodrug” refers to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of the present invention. Typical examples of prodrugs includecompounds that have biologically labile protecting groups on afunctional moiety of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated, dephosphorylated,thiophoshoramidated, dethiophoshoramidated, phoshoramidated ordephosphoramidated to produce the active compound. The compounds of thisinvention possess antiviral activity against HCV, or are metabolized toa compound that exhibits such activity. Theβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleoside can also be administered as a 5′-phosphoether lipid, abisphosphoramidate, a 3′,5′-cyclic phosphoramidate, a 3′,5′-cyclicthiophosphoramidate, a DTE conjugate, a mixed phosphoramidate-SATEderivative or a “SATE” derivative.

The term “phosphonic acid” refers to the group —P(O)(OH)₂.

In one embodiment, the term purine or pyrimidine base includes, but isnot limited to, adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acylis —C(O)alkyl, —C(O)(aryl)C₀-C₄alkyl, or —C(O)(C₀-C₄alkyl)aryl),N⁶-benzylpurine, N⁶-halopurine, N⁶-vinylpurine, N⁶-acetylenic purine,N⁶-acyl purine, N⁶-hydroxyalkyl purine, N⁶-thioalkyl purine,N²-alkylpurines, N²-alkyl-6-thiopurines, thymine, cytosine,5-fluorocytosine, 5-methylcytosine, 6-azapyrimidine, including6-azacytosine, 2- and/or 4-mercaptopyrmidine, uracil, 5-halouracil,including 5-fluorouracil, C⁵-alkylpyrimidines, C⁵-benzylpyrimidines,C⁵-halopyrimidines, C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine,C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine,C⁵-cyanopyrimidine, C⁵-nitropyrimidine, C⁵-aminopyrimidine,N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, andpyrazolo-pyrimidinyl. Purine bases include, but are not limited to,guanine, adenine, hypoxanthine, 2,6-diaminopurine, and 6-chloropurine.Functional oxygen and nitrogen groups on the base can be protected asnecessary or desired. Suitable protecting groups are well known to thoseskilled in the art, and include benzyl, trimethylsilyl,dimethylhexylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, trityl,alkyl groups, and acyl groups such as acetyl and propionyl;methanesulfonyl, and p-toluenesulfonyl. Alternatively, the purine orpyrimidine base can optionally be substituted such that it forms aviable prodrug, which can be cleaved in vivo. Examples of appropriatesubstituents include an acyl moiety.

The term “substituted” or “optionally substituted” indicates that themoiety can have at least one additional substituent including, but notlimited to, halogen (F, Cl, Br, I), OH, phenyl, benzyl, N₃, CN, acyl,alkyl, including methyl; alkenyl, alkynyl, alkoxy, haloalkyl; includingCHF₂, CH₂F and CF₃; etc. In one embodiment, the term “substituted” or“optionally substituted” indicates that the moiety can have at least oneadditional substituent including, but not limited to, azido, cyano,halogen (fluoro, chloro, bromo, or iodo), alkyl, alkenyl, alkynyl,cycloalkyl, heterocycle, aryl, heteroaryl, haloalkyl, hydroxyl, alkoxy,amino, —NH(C₁-C₆ unsubstituted alkyl), —NH(C₁-C₆ substituted alkyl),—NH—(C₀-C₂alkyl)(C₃-C₈cycloalkyl), —NH—(C₀-C₂alkyl)(C₃-C₈heterocycle),—NH—(C₀-C₂alkyl)(aryl), —N(C₁-C₆ unsubstituted alkyl)₂, —N(C₁-C₆unsubstituted alkyl)(C₁-C₆ substituted alkyl), —N(C₁-C₆ substitutedalkyl)₂, —NH—(C₀-C₂alkyl)(C₃-C₈cycloalkyl),—NH—(C₀-C₂alkyl)(C₃-C₈heterocycle), —NH—(C₀-C₂alkyl)(aryl), acyl, nitro,sulfonic acid, sulfate, phosphonic acid, phosphate, phosphonate, orthiol.

The term “sulfonate esters”, represented by the formula, R¹⁴S(O)₂OR¹⁵,comprise R¹⁴ wherein R¹⁴ is alkyl, haloalkyl, aralkyl or aryl. R¹⁵ isalkyl, aryl or aralkyl.

The term “sulfonic acid” refers to the group —SO₂OH.

The term “thiol” refers to the group —SH.

The term “nitrogen-protecting group” as used herein refers to a moietythat is covalently attached to nitrogen and which can be removed, andtypically replaced with hydrogen, when appropriate. For example, anitrogen-protecting group may be a group that is removed in vivo afteradministration to a host, in vitro by a cell, or it may be removedduring a manufacturing process. Suitable nitrogen-protecting groupsuseful in the present invention are described by Greene and Wuts inProtective Groups in Organic Synthesis (1991) New York, John Wiley andSons, Inc.

The term “oxygen-protecting group” as used herein refers to a moietythat is covalently attached to oxygen and which can be removed, andtypically replaced with hydrogen, when appropriate. For example, anoxygen-protecting group may be a group that is removed in vivo afteradministration to a host, in vitro by a cell, or it may be removedduring a manufacturing process. Suitable oxygen-protecting groups usefulin the present invention are described by Greene and Wuts in ProtectiveGroups in Organic Synthesis (1991) New York, John Wiley and Sons, Inc.

“Phosphate” refers to the group —OP(O)(OH)₂.

“Phosphate ester” refers to mono, di, and tri phosphates unlessotherwise indicated.

The term “phosphoamidate”, “phosphoramidate”, or “phosphoroamidate” is amoiety that has a phosphorus bound to three oxygen groups and an amine(which may optionally be substituted). Suitable phosphoramidates usefulin the present invention are described by Madela, Karolina and McGuiganin 2012, “Progress in the development of anti-hepatitis C virusnucleoside and nucleotide prodrugs”, Future Medicinal Chemistry 4(5),pages 625-650 10:1021/jm300074y and Dominique, McGuigan and Balzarini in2004, “Aryloxy Phosphoramidate Triesters as Pro-Tides”, Mini Reviews inMedicinal Chemistry 4(4), pages 371-381. Additional phosphoramidatesuseful in the present invention are described in U.S. Pat. Nos.5,233,031, 7,115,590, 7,547,704, 7,879,815, 7,888,330, 7,902,202,7,951,789, 7,964,580, 8,071,568; 8,148,349, 8,263,575, 8,324,179,8,334,270, 8,552,021, 8,563,530, 8,580,765, 8,735,372, 8,759,318; EP2120565; EP 1143995; U.S. Pat. Nos. 6,455,513; and 8,334,270. Otherphosphoramidates are described in the nucleoside patents described inthe Background of the Invention.

Phosphoramidate groups for use in the present invention include those ofthe structures:

Other phosphoramidates for use in the present invention include those ofthe structure:

-   -   wherein:    -   R^(P1) is an optionally substituted linear, branched, or cyclic        alkyl group, or an optionally substituted aryl, heteroaryl or        heterocyclic group or a linked combination thereof; and    -   R^(P2) is a —NR^(N1)R^(N2) group or a B′ group;    -   wherein:    -   R^(N1) and R^(N2) are each independently H, C₁₋₈alkyl,        (C₃-C₇cycloalkyl)C₀-C₄alkyl-, (aryl)C₀-C₄alkyl-,        (C₃-C₆heterocycle)C₀-C₄alkyl-, or (heteroaryl)C₀-C₄alkyl-; which        may be optionally substituted; or    -   R^(N1) and R^(N2) along with the nitrogen atom to which that are        attached, join to form a 3 to 7 membered heterocyclic ring;    -   B is a

group;

-   -   wherein:    -   R¹⁶ is hydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        (C₃-C₈cycloalkyl)C₀-C₄alkyl-, (aryl)C₀-C₄alkyl-,        (C₃-C₆heterocyclo)C₀-C₄alkyl-, (heteroaryl)C₀-C₄alkyl-, or the        sidechain of an amino acid, for example a sidechain of an amino        acid (as otherwise described herein) often selected from the        group consisting of alanine, β-alanine, arginine, asparagine,        aspartic acid, cysteine, cysteine, glutamic acid, glutamine,        glycine, phenylalanine, histidine, isoleucine, lysine, leucine,        methionine, proline, serine, threonine, valine, tryptophan, or        tyrosine (often R¹⁶ is hydrogen, methyl, isopropyl, or        isobutyl);    -   R¹⁷ is hydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        (C₃-C₈cycloalkyl)C₀-C₄alkyl-, (aryl)C₀-C₄alkyl-,        (C₃-C₆heterocyclo)C₀-C₄alkyl-, (heteroaryl)C₀-C₄alkyl-, or the        sidechain of an amino acid, for example a sidechain of an amino        acid (as otherwise described herein) often selected from the        group consisting of alanine, β-alanine, arginine, asparagine,        aspartic acid, cysteine, cysteine, glutamic acid, glutamine,        glycine, phenylalanine, histidine, isoleucine, lysine, leucine,        methionine, proline, serine, threonine, valine, tryptophan, or        tyrosine (often R¹⁷ is hydrogen, methyl, isopropyl, or        isobutyl);    -   R¹⁸ is hydrogen or C₁-C₃alkyl; or    -   R¹⁶ and R¹⁷ can form a (C₃-C₇)cycloalkyl or (C₃-C₇)heterocyclic        group; or    -   R¹⁸ and R¹⁶ or R¹⁷ can form (C₃-C₆)heterocyclic group; and    -   R¹⁹ is hydrogen, (C₁-C₆)alkyl, (C₃-C₆)alkenyl, (C₃-C₆)alkynyl,        (C₃-C₈cycloalkyl)C₀-C₄alkyl-, (aryl)C₀-C₄alkyl-,        (C₃-C₆heterocyclo)C₀-C₄alkyl-, (heteroaryl)C₀-C₄alkyl-; or    -   B′ is a

group;

-   -   wherein:    -   R²⁰ is hydrogen, (C₁-C₃)alkyl, (C₃-C₈cycloalkyl)C₀-C₄alkyl-,        (aryl)C₀-C₄alkyl-, (C₃-C₆heterocyclo)C₀-C₄alkyl-, or        (heteroaryl)C₀-C₄alkyl-;    -   R²¹ is hydrogen, (C₁-C₃)alkyl, (C₃-C₈cycloalkyl)C₀-C₄alkyl-,        (aryl)C₀-C₄alkyl-, (C₃-C₆heterocyclo)C₀-C₄alkyl-, or        (heteroaryl)C₀-C₄alkyl-; and    -   R¹⁸ and R¹⁹ are as defined above.

Preferred R^(P1) groups include optionally substituted phenyl, naphthyl,and monocyclic heteroaryl groups, especially those groups (particularlylipophilic groups) which enhance bioavailability of the compounds in thecells of the patient and which exhibit reduced toxicity, enhancedtherapeutic index and enhanced pharmacokinetics (the compounds aremetabolized and excreted more slowly).

The term phosphoramidate is used throughout the specification todescribe a group that is found at the 5′ or 3′ position of the furanosering of the nucleoside compound and forms a prodrug form of thenucleoside compound. In one embodiment, phosphoramidates can be found atboth the 5′ and 3′ position of the furanose ring of the nucleosidecompound and form a prodrug form of the nucleoside compound. In anotherembodiment, the phosphoramidate found at the 5′ position of the furanosering of the nucleoside can form a cyclic phosphoramidate compound byforming a bond with the 3′-hydroxyl substituent at the 3′ position ofthe furanose ring of the nucleoside compound and form a prodrug form ofthe nucleoside compound.

The term “thiophosphoamidate”, “thiophosphoramidate”, or“thiophosphoroamidate” is a moiety that has a phosphorus bound tosulfur, two oxygen groups and an amine (which may optionally besubstituted). Thiophosphoramidates useful in the present invention aredescribed in U.S. Pat. No. 8,772,474 and WO 2012/040124.

Thiophosphoramidate groups for use in the present invention includethose of the structures:

Other thiophosphoramidates include those of the structure:

-   -   wherein:    -   R^(P1) is an optionally substituted linear, branched, or cyclic        alkyl group, or an optionally substituted aryl, heteroaryl or        heterocyclic group or a linked combination thereof; and    -   R^(P2) is a —R^(N1)R^(N2) group or a B′ group;    -   wherein:    -   R^(N1) and R^(N2) are each independently H, C₁-C₈ alkyl,        (C₃-C₇cycloalkyl)C₀-C₄alkyl-, (aryl)C₀-C₄alkyl-,        (C₃-C₆heterocyclo)C₀-C₄alkyl-, or (heteroaryl)C₀-C₄alkyl-; or    -   R^(N1) and R^(N2) along with the nitrogen atom to which that are        attached, join to form a 3 to 7 membered heterocyclic ring;    -   B′ is a

group;

-   -   wherein:    -   R¹⁶ is hydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        (C₃-C₈cycloalkyl)C₀-C₄alkyl-, (aryl)C₀-C₄alkyl-,        (C₃-C₆heterocyclo)C₀-C₄alkyl-, (heteroaryl)C₀-C₄alkyl-, or the        sidechain of an amino acid, for example a sidechain of an amino        acid (as otherwise described herein) often selected from the        group consisting of alanine, β-alanine, arginine, asparagine,        aspartic acid, cysteine, cysteine, glutamic acid, glutamine,        glycine, phenylalanine, histidine, isoleucine, lysine, leucine,        methionine, proline, serine, threonine, valine, tryptophan, or        tyrosine (often R¹⁶ is hydrogen, methyl, isopropyl, or        isobutyl);    -   R¹⁷ is hydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,        (C₃-C₈cycloalkyl)C₀-C₄alkyl-, (aryl)C₀-C₄alkyl-,        (C₃-C₆heterocyclo)C₀-C₄alkyl-, (heteroaryl)C₀-C₄alkyl-, or the        sidechain of an amino acid, for example a sidechain of an amino        acid (as otherwise described herein) often selected from the        group consisting of alanine, β-alanine, arginine, asparagine,        aspartic acid, cysteine, cysteine, glutamic acid, glutamine,        glycine, phenylalanine, histidine, isoleucine, lysine, leucine,        methionine, proline, serine, threonine, valine, tryptophan, or        tyrosine (often R¹⁷ is hydrogen, methyl, isopropyl, or        isobutyl);    -   R¹⁸ is hydrogen or C₁-C₃alkyl; or    -   R¹⁶ and R¹⁷ can form a (C₃-C₇)cycloalkyl or (C₃-C₇)heterocyclic        group; or    -   R¹⁸ and R¹⁶ or R¹⁷ can form (C₃-C₆) heterocyclic group; and    -   R¹⁹ is hydrogen, (C₁-C₆)alkyl, (C₃-C₆)alkenyl, (C₃-C₆)alkynyl,        (C₃-C₈cycloalkyl)C₀-C₄alkyl-, (aryl)C₀-C₄alkyl-,        (C₃-C₆heterocyclo)C₀-C₄alkyl-, (heteroaryl)C₀-C₄alkyl-; or    -   B′ is a

group; and

-   -   R¹⁸, R¹⁹, R²⁰ and R²¹ are as defined above.

Preferred R^(P1) groups include optionally substituted phenyl, naphthyl,and monocyclic heteroaryl groups, especially those groups (particularlylipophilic groups) which enhance bioavailability of the compounds intothe cells of the patient and which exhibit reduced toxicity, enhancedtherapeutic index and enhanced pharmacokinetics (the compounds aremetabolized and excreted more slowly).

The thiophosphoramidate can be at the 5′ or 3′ position of the furanosering of the nucleoside compound to form a prodrug form of the nucleosidecompound. In one embodiment, thiophosphoramidates can be found at boththe 5′ and 3′ position of the furanose ring of the nucleoside compoundand form a prodrug form of the nucleoside compound. In anotherembodiment, the thiophosphoramidate found at the 5′ position of thefuranose ring of the nucleoside can form a cyclic thiophosphoramidatecompound by forming a bond with the 3′-hydroxyl substituent at the 3′position of the furanose ring of the nucleoside compound and form aprodrug form of the nucleoside compound.

The term “D-configuration” as used in the context of the presentinvention refers to the principle configuration which mimics the naturalconfiguration of sugar moieties as opposed to the unnatural occurringnucleosides or “L” configuration. The term “β” or “β anomer” is usedwith reference to nucleoside analogs in which the nucleoside base isconfigured (disposed) above the plane of the furanose moiety in thenucleoside analog.

The terms “coadminister” and “coadministration” or combination therapyare used to describe the administration of at least one of the2′-deoxy-2′-α-fluoro-2′-β-C-nucleoside compounds according to thepresent invention in combination with at least one other active agent,for example where appropriate at least one additional anti-HCV agent,including other 2′-deoxy-2′-α-fluoro-2′-β-C-nucleoside agents which aredisclosed herein. The timing of the coadministration is best determinedby the medical specialist treating the patient. It is sometimespreferred that the agents be administered at the same time.Alternatively, the drugs selected for combination therapy may beadministered at different times to the patient. Of course, when morethan one viral or other infection or other condition is present, thepresent compounds may be combined with other agents to treat that otherinfection or condition as required.

The term “host”, as used herein, refers to a unicellular ormulticellular organism in which a HCV virus can replicate, includingcell lines and animals, and typically a human. The term hostspecifically refers to infected cells, cells transfected with all orpart of a HCV genome, and animals, in particular, primates (includingchimpanzees) and humans. In most animal applications of the presentinvention, the host is a human patient. Veterinary applications, incertain indications, however, are clearly anticipated by the presentinvention (such as chimpanzees). The host can be for example, bovine,equine, avian, canine, feline, etc.

Isotopic Substitution

The present invention includes compounds and the use of compounds withdesired isotopic substitutions of atoms, at amounts above the naturalabundance of the isotope, i.e., enriched. Isotopes are atoms having thesame atomic number but different mass numbers, i.e., the same number ofprotons but a different number of neutrons. By way of general exampleand without limitation, isotopes of hydrogen, for example, deuterium(²H) and tritium (³H) may be used anywhere in described structures.Alternatively or in addition, isotopes of carbon, e.g., ¹³C and ¹⁴C, maybe used. A preferred isotopic substitution is deuterium for hydrogen atone or more locations on the molecule to improve the performance of thedrug. The deuterium can be bound in a location of bond breakage duringmetabolism (an α-deuterium kinetic isotope effect) or next to or nearthe site of bond breakage (a β-deuterium kinetic isotope effect).Achillion Pharmaceuticals, Inc. (WO/2014/169278 and WO/2014/169280)describes deuteration of nucleotides to improve their pharmacokineticsor pharmacodynamics, including at the 5-position of the molecule.

Substitution with isotopes such as deuterium can afford certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or reduced dosagerequirements. Substitution of deuterium for hydrogen at a site ofmetabolic break down can reduce the rate of or eliminate the metabolismat that bond. At any position of the compound that a hydrogen atom maybe present, the hydrogen atom can be any isotope of hydrogen, includingprotium (¹H), deuterium (²H) and tritium (³H). Thus, reference herein toa compound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

The term “isotopically-labeled” analog refers to an analog that is a“deuterated analog”, a “¹³C-labeled analog,” or a“deuterated/¹³C-labeled analog.” The term “deuterated analog” means acompound described herein, whereby a H-isotope, i.e., hydrogen/protium(¹H), is substituted by a H-isotope, i.e., deuterium (²H). Deuteriumsubstitution can be partial or complete. Partial deuterium substitutionmeans that at least one hydrogen is substituted by at least onedeuterium. In certain embodiments, the isotope is 90, 95 or 99% or moreenriched in an isotope at any location of interest. In some embodimentsit is deuterium that is 90, 95 or 99% enriched at a desired location.Unless indicated to the contrary, the deuteration is at least 80% at theselected location. Deuteration of the nucleoside can occur at anyreplaceable hydrogen that provides the desired results.

III. Methods of Treatment or Prophylaxis

Treatment, as used herein, refers to the administration of an activecompound to a host that is infected with a HCV virus.

The term “prophylactic” or preventative, when used, refers to theadministration of an active compound to prevent or reduce the likelihoodof an occurrence of the viral disorder. The present invention includesboth treatment and prophylactic or preventative therapies. In oneembodiment, the active compound is administered to a host who has beenexposed to and thus at risk of infection by a hepatitis C virusinfection.

The invention is directed to a method of treatment or prophylaxis of ahepatitis C virus, including drug resistant and multidrug resistantforms of HCV and related disease states, conditions, or complications ofan HCV infection, including cirrhosis and related hepatotoxicities, aswell as other conditions that are secondary to a HCV infection, such asweakness, loss of appetite, weight loss, breast enlargement (especiallyin men), rash (especially on the palms), difficulty with clotting ofblood, spider-like blood vessels on the skin, confusion, coma(encephalopathy), buildup of fluid in the abdominal cavity (ascites),esophageal varices, portal hypertension, kidney failure, enlargedspleen, decrease in blood cells, anemia, thrombocytopenia, jaundice, andhepatocellular cancer, among others. The method comprises administeringto a host in need thereof an effective amount of at least one0-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotide as described herein, optionally in combination with at leastone additional bioactive agent, for example, an additional anti-HCVagent, further in combination with a pharmaceutically acceptable carrieradditive and/or excipient.

In yet another aspect, the present invention is a method for preventionor prophylaxis of a an HCV infection or a disease state or related orfollow-on disease state, condition or complication of an HCV infection,including cirrhosis and related hepatotoxicities, weakness, loss ofappetite, weight loss, breast enlargement (especially in men), rash(especially on the palms), difficulty with clotting of blood,spider-like blood vessels on the skin, confusion, coma (encephalopathy),buildup of fluid in the abdominal cavity (ascites), esophageal varices,portal hypertension, kidney failure, enlarged spleen, decrease in bloodcells, anemia, thrombocytopenia, jaundice, and hepatocellular (liver)cancer, among others, said method comprising administering to a patientat risk with an effective amount of at least one compound according tothe present invention as described above in combination with apharmaceutically acceptable carrier, additive, or excipient, optionallyin combination with another anti-HCV agent. In another embodiment, theactive compounds of the invention can be administered to a patient aftera hepatitis-related liver transplantation to protect the new organ.

The 5′-stabilizedβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotide can be administered if desired as any salt or prodrug thatupon administration to the recipient is capable of providing directly orindirectly the parent compound, or that exhibits activity itself.Nonlimiting examples are the pharmaceutically acceptable salts and acompound, which has been modified at a function group, such as ahydroxyl or amine function, to modify the biological activity,pharmacokinetics, half-life, controlled delivery, lipophilicity,absorption kinetics, ease of phosphorylation to the active5′-triphosphate or efficiency of delivery using a desired route ofadministration of the compound. Methods to modify the properties of anactive compound to achieve target properties are known to those of skillin the art or can easily be assessed by standard methods, for example,acylation, phosphorylation, thiophosphoramidation, phosphoramidation,phosphonation, alkylation, or pegylation.

IV. Pharmaceutical Compositions

In an aspect of the invention, pharmaceutical compositions according tothe present invention comprise an anti-HCV virus effective amount of atleast one of the 5′-stabilizedβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotide compounds described herein, optionally in combination with apharmaceutically acceptable carrier, additive, or excipient, furtheroptionally in combination or alternation with at least one other activecompound.

In an aspect of the invention, pharmaceutical compositions according tothe present invention comprise an anti-HCV effective amount of at leastone of the activeβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotide compounds described herein, optionally in combination with apharmaceutically acceptable carrier, additive, or excipient, furtheroptionally in combination with at least one other antiviral, such as ananti-HCV agent.

The invention includes pharmaceutical compositions that include aneffective amount to treat a hepatitis C virus infection, of one of theβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotide compounds of the present invention or its salt or prodrug, ina pharmaceutically acceptable carrier or excipient. In an alternativeembodiment, the invention includes pharmaceutical compositions thatinclude an effective amount to prevent a hepatitis C virus infection, ofone of theβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotide compounds of the present invention or its salt or prodrug, ina pharmaceutically acceptable carrier or excipient.

One of ordinary skill in the art will recognize that a therapeuticallyeffective amount will vary with the infection or condition to betreated, its severity, the treatment regimen to be employed, thepharmacokinetics of the agent used, as well as the patient or subject(animal or human) to be treated, and such therapeutic amount can bedetermined by the attending physician or specialist.

The 5′-stabilized β-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified—N⁶-substituted purine nucleotide compounds according to the presentinvention can be formulated in an admixture with a pharmaceuticallyacceptable carrier. In general, it is preferable to administer thepharmaceutical composition in orally-administrable form, but certainformulations may be administered via a parenteral, intravenous,intramuscular, topical, transdermal, buccal, subcutaneous, suppository,or other route, including intranasal spray. Intravenous andintramuscular formulations are often administered in sterile saline. Oneof ordinary skill in the art may modify the formulations to render themmore soluble in water or other vehicle, for example, this can be easilyaccomplished by minor modifications (salt formulation, esterification,etc.) which are well within the ordinary skill in the art. It is alsowell within the routineers' skill to modify the route of administrationand dosage regimen of a particular compound in order to manage thepharmacokinetics of the present compounds for maximum beneficial effectin patients.

In certain pharmaceutical dosage forms, the prodrug form of thecompounds, especially including acylated (acetylated or other), andether (alkyl and related) derivatives, phosphate esters,thiophosphoramidates, phosphoramidates, and various salt forms of thepresent compounds, are preferred. One of ordinary skill in the art willrecognize how to readily modify the present compounds to prodrug formsto facilitate delivery of active compounds to a targeted site within thehost organism or patient. The routineer also will take advantage offavorable pharmacokinetic parameters of the prodrug forms, whereapplicable, in delivering the present compounds to a targeted sitewithin the host organism or patient to maximize the intended effect ofthe compound.

The amount of compound included within therapeutically activeformulations according to the present invention is an effective amountfor treating the HCV infection, reducing the likelihood of a HCVinfection or the inhibition, reduction, and/or abolition of HCV or itssecondary effects, including disease states, conditions, and/orcomplications which occur secondary to HCV. In general, atherapeutically effective amount of the present compound inpharmaceutical dosage form usually ranges from about 0.001 mg/kg toabout 100 mg/kg per day or more, more often, slightly less than about0.1 mg/kg to more than about 25 mg/kg per day of the patient orconsiderably more, depending upon the compound used, the condition orinfection treated and the route of administration. The active nucleosidecompound according to the present invention is often administered inamounts ranging from about 0.1 mg/kg to about 15 mg/kg per day of thepatient, depending upon the pharmacokinetics of the agent in thepatient. This dosage range generally produces effective blood levelconcentrations of active compound which may range from about 0.001 toabout 100, about 0.05 to about 100 micrograms/cc of blood in thepatient.

Often, to treat, prevent or delay the onset of these infections and/orto reduce the likelihood of an HCV virus infection, or a secondarydisease state, condition or complication of HCV, the compositions willbe administered in oral dosage form in amounts ranging from about 250micrograms up to about 500 mg or more at least once a day, for example,at least 25, 50, 100, 150, 250 or 500 milligrams, up to four times aday. The present compounds are often administered orally, but may beadministered parenterally, topically, or in suppository form, as well asintranasally, as a nasal spray or as otherwise described herein.

In the case of the co-administration of the present compounds incombination with another anti-HCV compound as otherwise describedherein, the amount of the compound according to the present invention tobe administered ranges from about 0.01 mg/kg of the patient to about 500mg/kg. or more of the patient or considerably more, depending upon thesecond agent to be co-administered and its potency against the virus,the condition of the patient and severity of the disease or infection tobe treated and the route of administration. The other anti-HCV agent mayfor example be administered in amounts ranging from about 0.01 mg/kg toabout 500 mg/kg. In certain preferred embodiments, these compounds maybe often administered in an amount ranging from about 0.5 mg/kg to about50 mg/kg or more (usually up to about 100 mg/kg), generally dependingupon the pharmacokinetics of the two agents in the patient. These dosageranges generally produce effective blood level concentrations of activecompound in the patient.

For purposes of the present invention, a prophylactically or preventiveeffective amount of the compositions according to the present inventionfalls within the same concentration range as set forth above fortherapeutically effective amount and is usually the same as atherapeutically effective amount.

Administration of the active compound may range from continuous(intravenous drip) to several oral or intranasal administrations per day(for example, Q.I.D.) or transdermal administration and may includeoral, topical, parenteral, intramuscular, intravenous, sub-cutaneous,transdermal (which may include a penetration enhancement agent), buccal,and suppository administration, among other routes of administration.Enteric coated oral tablets may also be used to enhance bioavailabilityof the compounds for an oral route of administration. The most effectivedosage form will depend upon the bioavailability/pharmacokinetics of theparticular agent chosen as well as the severity of disease in thepatient. Oral dosage forms are particularly preferred, because of easeof administration and prospective favorable patient compliance.

To prepare the pharmaceutical compositions according to the presentinvention, a therapeutically effective amount of one or more of thecompounds according to the present invention is often intimately admixedwith a pharmaceutically acceptable carrier according to conventionalpharmaceutical compounding techniques to produce a dose. A carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, e.g., oral or parenteral. In preparingpharmaceutical compositions in oral dosage form, any of the usualpharmaceutical media may be used. Thus, for liquid oral preparationssuch as suspensions, elixirs, and solutions, suitable carriers andadditives including water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like may be used. For solid oralpreparations such as powders, tablets, capsules, and for solidpreparations such as suppositories, suitable carriers and additivesincluding starches, sugar carriers, such as dextrose, manifold, lactose,and related carriers, diluents, granulating agents, lubricants, binders,disintegrating agents, and the like may be used. If desired, the tabletsor capsules may be enteric-coated or sustained release by standardtechniques. The use of these dosage forms may significantly enhance thebioavailability of the compounds in the patient.

For parenteral formulations, the carrier will usually comprise sterilewater or aqueous sodium chloride solution, though other ingredients,including those which aid dispersion, also may be included. Of course,where sterile water is to be used and maintained as sterile, thecompositions and carriers must also be sterilized. Injectablesuspensions may also be prepared, in which case appropriate liquidcarriers, suspending agents, and the like may be employed.

Liposomal suspensions (including liposomes targeted to viral antigens)may also be prepared by conventional methods to produce pharmaceuticallyacceptable carriers. This may be appropriate for the delivery of freenucleosides, acyl/alkyl nucleosides or phosphate ester prodrug forms ofthe nucleoside compounds according to the present invention.

In typical embodiments according to the present invention, the compoundsand compositions are used to treat, prevent or delay a HCV infection ora secondary disease state, condition or complication of HCV.

V. Combination and Alternation Therapy

It is well recognized that drug-resistant variants of viruses can emergeafter prolonged treatment with an antiviral agent. Drug resistance mosttypically occurs by mutation of a gene that encodes for an enzyme usedin viral replication. The efficacy of a drug against an HCV infection,can be prolonged, augmented, or restored by administering the compoundin combination or alternation with another, and perhaps even two orthree other, antiviral compounds that induce a different mutation or actthrough a different pathway, from that of the principle drug.Alternatively, the pharmacokinetics, bio distribution, half-life, orother parameter of the drug can be altered by such combination therapy(which may include alternation therapy if considered concerted). Sincethe disclosedβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotides are NS5B polymerase inhibitors, it may be useful toadminister the compound to a host in combination with, for example a:

-   -   (1) Protease inhibitor, such as an NS3/4A protease inhibitor;    -   (2) NS5A inhibitor;    -   (3) Another NS5B polymerase inhibitor;    -   (4) NS5B non-substrate inhibitor;    -   (5) Interferon alfa-2a, which may be pegylated or otherwise        modified, and/or ribavirin;    -   (6) Non-substrate-based inhibitor;    -   (7) Helicase inhibitor;    -   (8) Antisense oligodeoxynucleotide (S-ODN);    -   (9) Aptamer;    -   (10) Nuclease-resistant ribozyme;    -   (11) iRNA, including microRNA and SiRNA;    -   (12) Antibody, partial antibody or domain antibody to the virus,        or    -   (13) Viral antigen or partial antigen that induces a host        antibody response.

Non limiting examples of anti-HCV agents that can be administered incombination with theβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substituted purinenucleotides of the invention are:

-   -   (i) protease inhibitors such as telaprevir (Incivek®),        boceprevir (Victrelis™), simeprevir (Olysio™), paritaprevir        (ABT-450), ACH-2684; AZD-7295; BMS-791325; danoprevir;        Filibuvir; GS-9256; GS-9451; MK-5172; Setrobuvir; Sovaprevir;        Tegobuvir; VX-135; VX-222 and ALS-220;    -   (ii) NS5A inhibitor such as ACH-2928, ACH-3102, IDX-719,        daclatasvir, ledispasvir and Ombitasvir (ABT-267);    -   (iii) NS5B inhibitors such as ACH-3422; AZD-7295; Clemizole;        ITX-5061; PPI-461; PPI-688, Sovaldi®, MK-3682, and mericitabine;    -   (iv) NS5B inhibitors such as ABT-333, MBX-700; and,    -   (v) Antibody such as GS-6624.

If the β-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substitutedpurine nucleotide is administered to treat advanced hepatitis C virusleading to liver cancer or cirrhosis, in one embodiment, the compoundcan be administered in combination or alternation with another drug thatis typically used to treat hepatocellular carcinoma (HCC), for example,as described by Andrew Zhu in “New Agents on the Horizon inHepatocellular Carcinoma” Therapeutic Advances in Medical Oncology, V5(1), January 2013, 41-50. Examples of suitable compounds forcombination therapy where the host has or is at risk of HCC includeanti-angiogenic agents, sunitinib, brivanib, linifanib, ramucirumab,bevacizumab, cediranib, pazopanib, TSU-68, lenvatinib, antibodiesagainst EGFR, mTor inhibitors, MEK inhibitors, and histone decetylaceinhibitors.

Drugs that are currently approved for influenza are Amantadine,Rimantadine and Oseltamivir. Any of these drugs can be used incombination or alternation with an active compound provided herein totreat a viral infection susceptible to such. Ribavirin is used to treatmeasles, Influenza A, influenza B, parainfluenza, severe RSVbronchiolitis and SARS as well as other viral infections, and thereforeis particularly useful in combination with the present compound fortreatment of the host infected with a single stranded RNA virus.Palivizumab is approved for use in infants with high risk for RSVinfection.

Currently, there are no approved drugs for West Nile virus. Physiciansare recommended to provide intensive support therapy, which may involvehospitalization, intravenous fluids, use of a ventilator to assistbreathing, medications to control seizures, brain swelling, nausea andvomiting, and the use of antibiotics to prevent bacterial infections formaking the disease even worse. This highlights the importance of thepresent compounds for viral medical therapy.

VI. Process of Preparation ofβ-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-Substituted PurineNucleotides of the Invention

General methods for providing the compounds of the present invention areknown in the art or described herein. The synthesis of 2′-chloronucleotides is described in US 20150366888, WO 2014058801; WO2015/066370 and WO 2015200219.

The following abbreviations are used in the synthetic schemes.

-   -   CBr₄: Carbon tetrabromide    -   DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene    -   DCM: Dichloromethane    -   THF: Tetrahydrofuran (THF), anhydrous    -   EtOAc: Ethyl acetate    -   EtOH: Ethanol    -   Li(OtBu)₃AlH: Lithium tri-tert-butoxyaluminum hydride    -   Na₂SO₄: Sodium sulphate (anhydrous)    -   MeCN: Acetonitrile    -   MeNH₂: Methylamine    -   MeOH: Methanol    -   Na₂SO₄: Sodium sulfate    -   NaHCO₃: Sodium bicarbonate    -   NH₄Cl: Ammonium chloride    -   NH₄OH: Ammonium hydroxide    -   PE: Petroleum ether    -   Ph₃P: Triphenylphosphine    -   Silica gel (230 to 400 mesh, Sorbent)    -   t-BuMgCl: t-Butyl magnesium chloride    -   t-BuOK: Sodium tert-butoxide    -   t-BuOH: Tert-butanol

EXAMPLES General Methods

¹H, ¹⁹F and ³¹P NMR spectra were recorded on a 300 MHz Fourier transformBrucker spectrometer. Spectra were obtained from samples prepared in 5mm diameter tubes in CDCl₃, CD₃OD or DMSO-d₆. The spin multiplicitiesare indicated by the symbols s (singlet), d (doublet), t (triplet), m(multiplet) and, br (broad). Coupling constants (J) are reported in Hz.MS spectra were obtained using electrospray ionization (ESI) on anAgilent Technologies 6120 quadrupole MS apparatus. The reactions weregenerally carried out under a dry nitrogen atmosphere usingSigma-Aldrich anhydrous solvents. All common chemicals were purchasedfrom commercial sources.

Example 1. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninateStep 1. Preparation of((2R,3R,4R,5R)-3-(benzoyloxy)-5-bromo-4-fluoro-4-methyltetrahydrofuran-2-yl)methylbenzoate(2)

To a solution of(2R)-3,5-di-1-benzoyl-2-fluoro-2-C-methyl-D-ribono-γ-lactone (24.8 g,66.6 mmol) in dry THE (333 mL), under a nitrogen atmosphere and cooledto −30° C., was added lithium tri-tert-butoxyaluminum hydride (1.0 M inTHF, 22.6 mL, 22.6 mmol) dropwise. After completion of the addition thereaction mixture was slowly warmed up to −15° C. over 90 min then EtOAcwas added (300 mL) and the mixture was quenched with a saturated aq.NH₄Cl solution (200 mL). The resulting solution was filtered on Celite®and the filtrate was extracted twice with EtOAc. The combined organicswere dried (Na₂SO₄), filtered and concentrated. The residue was taken upin dry DCM (225 mL) under a nitrogen atmosphere, cooled to −20° C., thenPPh₃ (19.1 g, 72.8 mmol) was added. After 10 min of stirring at −20° C.,CBr₄ (26.0 g, 78.4 mmol) was added and the reaction mixture was allowedto slowly warm up to 0° C. over 2 h. The resulting mixture was pouredonto a silica gel column and eluted with PE/EtOAc (gradient 100:0 to80:20). The fractions containing the α-bromofuranoside were collectedand concentrated to afford the product 2 (18.1 g, 41.3 mmol, 62% overtwo steps) as a thick colorless oil.

¹H NMR (300 MHz, CDCl₃) δ 8.15-8.11 (m, 2H), 8.04-8.01 (m, 2H),7.64-7.55 (m, 2H), 7.51-7.41 (m, 4H), 6.34 (d, J=1.6 Hz, 1H), 5.29 (dd,J=5.5, 3.1 Hz, 1H), 4.89-4.85 (m, 1H), 4.78 (dd, J=12.5, 3.2 Hz, 1H),4.63 (dd, J=12.5, 4.5 Hz, 1H), 1.72 (d, J=21.6 Hz, 3H). ¹⁹F NMR (282MHz, CDCl₃) δ −150.0.

Step 2. Preparation of(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate(3)

2-Amino-6-chloropurine (2.63 g, 15.5 mmol) was suspended in t-BuOH (54mL) under a nitrogen atmosphere. The reaction mixture was heated to 30°C. then potassium tert-butoxide (1.69 g, 15.1 mmol) was added. After 45min a solution of bromofuranoside 2 (2.24 g, 5.12 mmol) dissolved inanhydrous MeCN (6 mL) was added, the reaction mixture was heated to 65°C. for 16 h then cooled down to room temperature. A saturated aq. NH₄Clsolution (70 mL) was added and the resulting solution was extracted withEtOAc (3×60 mL). The combined organics were dried (Na₂SO₄), filtered andconcentrated. The residue was purified twice by column chromatography(gradient PE/EtOAc 80:20 to 0:100 then 60:40 to 20:80) to afford theproduct 3 (1.56 g, 2.96 mmol, 57%) as a white solid.

¹H NMR (300 MHz, CDCl₃) δ 8.05-8.02 (m, 2H), 7.95-7.92 (m, 2H), 7.88 (s,1H), 7.63-7.57 (m, 1H), 7.53-7.41 (m, 3H), 7.35-7.30 (m, 2H), 6.43 (dd,J=22.6, 9.1 Hz, 1H), 6.12 (d, J=18.3 Hz, 1H), 5.34 (br s, 2H), 5.00 (dd,J=11.9, 4.5 Hz, 1H), 4.79-4.73 (m, 1H), 4.60 (dd, J=11.9, 5.3 Hz, 1H),1.34 (d, J=22.6 Hz, 3H). ¹⁹F NMR (282 MHz, CDCl₃) δ −157.0. MS (ESI) m/zcalcd. for C₂₅H₂₂FN₅O₅ [M+H]⁺ 526.9; found 527.0.

Step 3. Preparation of(2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(4)

To a solution of compound 3 (575 mg, 1.09 mmol) in MeOH (9 mL) was addedmethylamine (33% in absolute EtOH, 1.7 mL, 1.81 mmol). The reactionmixture was heated to 85° C. in a sealed tube for 16 h, cooled down toroom temperature and concentrated. The residue was purified by columnchromatography (gradient DCM/MeOH 100:0 to 85:15) then reverse phasecolumn chromatography (gradient H₂O/MeOH 100:0 to 0:100) to afford theproduct 4 (286 mg, 0.91 mmol, 84%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 8.06 (s, 1H), 6.11 (d, J=18.1 Hz, 1H), 4.41(dd, J=24.4, 9.1 Hz, 1H), 4.07-4.01 (m, 2H), 3.86 (dd, J=12.9, 3.3 Hz,1H), 3.04 (br s, 3H), 1.16 (d, J=22.3 Hz, 3H). ¹⁹F NMR (282 MHz, CD₃OD)δ −163.7. MS (ESI) m/z calcd. for C₁₂H₁₉FN₆O₃ [M+H]⁺ 313.1; found 313.2.

Step 4. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(5)

To a solution of compound 4 (114 mg, 365 μmol) in dry THE (4 mL), undera nitrogen atmosphere and cooled to 0° C. was added t-butyl magnesiumchloride (1.0 M in THF, 0.66 mL, 660 μmol) dropwise over 10 min. Thereaction mixture was stirred 15 min at 0° C. then another 15 min at roomtemperature. The reaction mixture was cooled down to 0° C. then asolution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate, Ross, B.S., Reddy, P. G., Zhang, H. R., Rachakonda, S., and Sofia, M. J., J.Org, Chem., (2011), (253 mg, 558 μmol) dissolved in dry THE (1 mL) wasadded dropwise over 10 min. The reaction mixture was stirred at 0° C.for 30 min followed by 18 h at room temperature then quenched with asaturated aq. NH₄Cl solution (4 mL) and extracted with EtOAc (3×5 mL).The combined organics were dried, filtered (Na₂SO₄) and concentrated.The residue was purified by column chromatography (gradient DCM/MeOH100:0 to 90:10) then reverse phase column chromatography (gradientH₂O/MeOH 100:0 to 0:100) to afford product 5 (a mixture ofdiastereomers, 101 mg, 174 μmol, 48%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 7.83 (s, 0.55H), 7.82 (s, 0.45H), 7.38-7.16(m, 5H), 6.15 (d, J=18.5 Hz, 0.45H), (d, J=18.8 Hz, 0.55H), 4.99-4.88(overlapped with H₂O, m, 1H), 4.65-4.36 (m, 3H), 4.25-4.17 (m, 1H),3.97-3.85 (m, 1H), 3.05 (br s, 3H), 1.32-1.28 (m, 3H), 1.25-1.15 (m,9H). ¹⁹F NMR (282 MHz, CD₃OD) δ −162.8 (s), −163.3 (s). 3P NMR (121 MHz,CD₃OD) δ 4.10 (s), 3.99 (s). MS (ESI) m/z calcd. for C₂₄H₃₄FN₇O₇P [M+H]⁺582.2; found 582.2.

Example 2. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-Amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(7) Step 1. Preparation of(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(6)

To a solution of compound 3, from Example 1, (500 mg, 0.95 mmol) in MeOH(6 mL) was added dimethylamine hydrochloride (783 mg, 9.6 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (1.43 mL, 9.6 mmol). The reactionmixture was heated at 85° C. in a sealed tube for 6 h, cooled down toroom temperature and concentrated. The residue was purified by columnchromatography (gradient DCM/MeOH 100:0 to 85:15) then by reverse phasecolumn chromatography (gradient H₂O/MeOH 100:0 to 0:100) to affordproduct 6 (200 mg, 0.61 mmol, 64%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 8.07 (s, 1H), 6.14 (d, J=18.1 Hz, 1H), 4.41(dd, J=24.4, 9.2 Hz, 1H), 4.08-4.02 (m, 2H), 3.87 (dd, J=12.8, 2.9 Hz,1H), 3.42 (br s, 6H), 1.16 (d, J=22.0 Hz, 3H). ¹⁹F NMR (282 MHz, CD₃OD)δ −163.8. MS (ESI) m/z calcd. for C₁₃H₂OFN₆O₃ [M+H]⁺ 327.2; found 327.2.

Step 2. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(7)

To a solution of compound 6 (80 mg, 245 μmol) in dry THE (4 mL), under anitrogen atmosphere and cooled to 0° C. was added tert-butyl magnesiumchloride (1.0 M in THF, 0.64 mL, 640 μmol) drop-wise over 10 min. Thereaction mixture was stirred 15 min at 0° C. then another 15 min at roomtemperature. The reaction mixture was cooled down to 0° C. then asolution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate (167 mg, 367μmol) dissolved in dry THE (4 mL) was added drop-wise over 10 min. Thereaction mixture was stirred at 0° C. for 30 min and 18 h at roomtemperature. The reaction was quenched with a saturated aq. NH₄Clsolution (4 mL) and extracted with EtOAc (3×5 mL). The combined organicswere dried, filtered (Na₂SO₄) and concentrated. The residue was purifiedby column chromatography (gradient DCM/MeOH 100:0 to 90:10) and then byreverse phase column chromatography (gradient H₂O/MeOH 100:0 to 0:100)to afford the product 7 (mixture of diastereomers, 35 mg, 58 μmol, 24%)as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 7.83 (s, 0.5H), 7.82 (s, 0.5H), 7.34-7.16 (m,5H), 6.15 (d, J=18.7 Hz, 0.5H), 6.13 (d, J=18.8 Hz, 0.5H), 4.99-4.85(overlapped with H₂O, m, 1H), 4.65-4.26 (m, 3H), 4.27-4.12 (m, 1H),3.99-3.81 (m, 1H), 3.42, 3.41 (2br s, 6H), 1.36-1.25 (m, 3H), 1.24-1.11(m, 9H). ¹⁹F NMR (282 MHz, CD₃OD) δ −162.7 (s), −163.2 (s). ³¹P NMR (121MHz, CD₃OD) δ 4.08 (s), 4.00 (s). MS (ESI) m/z calcd. for C₂₅H₃₆FN₇O₇P[M+H]⁺ 596.5; found 596.2.

Example 3. Preparation of Isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-cyclopropylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(9) Step 1. Preparation of(2R,3R,4R,5R)-5-(2-Amino-6-(N-methyl-cyclopropylamino)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(8)

To a solution of compound 3 (600 mg, 1.14 mmol) in MeOH (10 mL) wasadded N-methylcyclopropylamine hydrochloride (366 mg, 3.40 mmol) andtriethylamine (470 μL, 3.40 mmol). The reaction mixture was heated at100° C. in a sealed tube for 15 h and cooled down to room temperature.An aqueous solution containing 30% NH₄OH (4 mL) was added and thereaction mixture was heated at 100° C. in a sealed tube for 2 h, cooleddown and concentrated. The residue was purified by column chromatography(gradient DCM/MeOH 100:0 to 90:10) to afford product 8 (351 mg, 0.99mmol, 87%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 8.13 (s, 1H), 6.15 (d, J=18.0 Hz, 1H), 4.40(dd, J=24.3, 9.0 Hz, 1H), 4.06-4.02 (m, 2H), 3.89-3.83 (m, 1H), 3.32 (m,3H), 3.18-3.11 (m, 1H), 1.16 (d, J=22.2 Hz, 3H), 0.96-0.89 (m, 2H),0.74-0.69 (m, 2H). ¹⁹F NMR (282 MHz, CD₃OD) δ −163.8. MS (ESI) m/zcalcd. for C₁₅H₂₂FN₆O₃ [M+H]⁺ 353.2; found 353.2.

Step 2. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-cyclopropylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(9)

To a solution of compound 8 (200 mg, 0.57 mmol) in dry THE (15 mL) at 0°C. was added tert-butyl magnesium chloride (1.0 M in THF, 680 μL, 0.68mmol) dropwise over 10 min. The reaction mixture was stirred 15 min at0° C. then another 15 min at room temperature. The reaction mixture wascooled down to 0° C. and a solution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate (283 mg,0.62 mmol) dissolved in dry THE (4 mL) was added dropwise over 10 min.The reaction mixture was stirred at 0° C. for 30 min and 18 h at roomtemperature. The reaction was quenched with a saturated aq. NH₄Clsolution (4 mL) and extracted with EtOAc (3×5 mL). The combined organicswere dried over Na₂SO₄ and concentrated. The residue was purified bycolumn chromatography (gradient DCM/MeOH 100:0 to 90:10) and then byreverse phase column chromatography (gradient H₂O/MeOH 100:0 to 0:100)to afford product 9 (mixture of 2 diastereoisomers, 160 mg, 0.26 mmol,45%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 7.85 (m, 1H), 7.38-7.16 (m, 5H), 6.18 (d,J=18.6 Hz) and 6.16 (d, J=18.9 Hz, 1H), 4.95-4.90 (overlapped with H₂O,m, 1H), 4.58-4.47 (m, 3H), 4.22-4.19 (m, 1H), 3.95-3.87 (m, 1H),3.36-3.34 (overlapped with MeOH, m, 3H), 3.19-3.12 (m, 1H), 1.32-1.22(m, 12H), 0.96-0.89 (m, 2H), 0.74-0.69 (m, 2H). ³¹P NMR (121 MHz, CD₃OD)δ 4.11 (s), 4.02 (s). MS (ESI) m/z calcd. for C₂₇H₃₈FN₇O₇P [M+H]⁺ 622.2;found 622.2.

Example 4. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2,6-bis-methylamino-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(12) Step 1. Preparation of(2R,3R,4R,5R)-5-(2,6-dichloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate (10)

The compound 2,6-dichloropurine (1.30 g, 6.86 mmol) was suspended int-BuOH (25 mL) under a nitrogen atmosphere. Potassium tert-butoxide (778mg, 6.92 mmol) was added portion-wise then the reaction mixture wasstirred at room temperature. After 1 h, a solution of bromofuranoside 2(1.0 g, 2.29 mmol) dissolved in anhydrous MeCN (20 mL) was added and thereaction mixture was heated at 65° C. overnight and then cooled down toroom temperature. A saturated aq. NH₄Cl solution was added and theresulting solution was extracted with EtOAc (3 times). The combinedorganics were dried over Na₂SO₄ and concentrated. The residue waspurified by column chromatography (gradient PE/EtOAc 100:0 to 0:100) toafford product 10 (148 mg, 0.27 mmol, 12%) as a sticky solid.

¹H NMR (300 MHz, CDCl₃) δ 8.31 (s, 1H), 8.12-8.09 (m, 2H), 8.02-7.99 (m,2H), 7.64-7.39 (m, 6H), 6.38 (d, J=17.2 Hz, 1H), 6.02 (dd, J=21.2, 8.9Hz, 1H), 4.90-4.68 (m, 3H), 1.33 (d, J=22.4 Hz, 3H). ¹⁹F NMR (282 MHz,CDCl₃) δ −158.0. MS (ESI) m/z calcd. for C₂₅H₂₀Cl₂FN₄O₅ [M+H]⁺ 546.4;found 546.3.

Step 2. Preparation of(2R,3R,4R,5R)-5-(2,6-bis-methylamino-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(11)

A solution of compound 10 (148 mg, 0.27 mmol) in methylamine (33% inEtOH, 30 mL) was heated at 130° C. in a sealed tube for 4 days, cooleddown to room temperature and concentrated. The residue was purified bycolumn chromatography (gradient DCM/MeOH 100:0 to 50:50) followed byreverse phase column chromatography (gradient H₂O/MeOH 100:0 to 0:100)to afford product 11 (33 mg, 0.10 mmol, 37%) as a white solid. ¹H NMR(300 MHz, CD₃OD) δ 8.00 (s, 1H), 6.12 (d, J=18.5 Hz, 1H), 4.51 (dd,J=24.4, 9.5 Hz, 1H), 4.06-3.85 (m, 3H), 3.04 (s, 3H), 2.93 (s, 3H), 1.20(d, J=22.4 Hz, 3H). ¹⁹F NMR (282 MHz, CD₃OD) δ −163.2. MS (ESI) m/zcalcd. for C₁₃H₂OFN₆O₃ [M+H]⁺ 327.2; found 327.2.

Step 3. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2,6-bis-methylamino-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(12)

To a solution of compound 11 (55 mg, 0.17 mmol) in dry THE (2 mL) at 0°C. was added tert-butyl magnesium chloride (1 M in THF, 304 DL, 0.30mmol) dropwise over 10 min. The reaction mixture was stirred 15 min at0° C. and then 15 min at room temperature. The solution was cooled downto 0° C. and a solution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate (115 mg,0.25 mmol) dissolved in dry THF (1 mL) was dropwise added over 10 min.The mixture was warmed slowly to room temperature and stirred for 4days. The reaction was quenched with a saturated aq. NH₄Cl solution andextracted with EtOAc (3 times). The combined organics were dried overNa₂SO₄ and concentrated. The residue was purified by columnchromatography (gradient DCM/MeOH 100:0 to 50:50) to yield product 12(mixture of diastereomers, 13 mg, 0.02 mmol, 13%) as a white solid. ¹HNMR (300 MHz, CD₃OD) δ 7.78 (s, 1H), 7.35-7.12 (m, 5H), 6.13 (d, J=19.1Hz, 0.53H), 6.10 (d, J=19.2 Hz, 0.47H), 4.99-4.78 (overlapped with H₂O,m, 1H), 4.72-4.46 (m, 3H), 4.24-4.15 (m, 1H), 3.79-3.92 (m, 1H), 3.02(br s, 3H), 2.92 (s+s, 3H), 1.29-1.11 (m, 12H). ¹⁹F NMR (282 MHz, CD₃OD)δ −162.0 (s), −162.3 (s). ³¹P NMR (121 MHz, CD₃OD) δ 3.97 (s), 3.89 (s).MS (ESI) m/z calcd. for C₂₅H₃₆FN₇O₇P [M+H]⁺ 596.6; found 596.2.

Example 5. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-isobutylamido-6-methylamino-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(16) Step 1. Preparation of Compound 13.

To a solution of compound 4 (286 mg, 0.92 mmol) and imidazole (370 mg,5.43 mmol) in dry DMF (6 mL) at 0° C. was added1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (300 μL, 0.94 mmol). Thereaction mixture was stirred for 2 h at RT, diluted with EtOAc (50 mL)and the suspension was washed with saturated aq. NH₄Cl solution andbrine (40 mL each). The organics were dried over Na₂SO₄ andconcentrated. The residue was purified by column chromatography(gradient PE/EtOAc 7:3 to 3:7) to afford product 13 (283 mg, 0.51 mmol,56%) as a white solid. MS (ESI) m/z calcd. for C₂₄H₄₄FN₆O₄Si₂ [M+H]⁺555.8; found 555.2.

Step 2. Preparation of compound 14.

To a solution of compound 13 (200 mg, 0.36 mmol) in dry pyridine (3 mL)at 0° C. was added isobutyryl chloride (38 μL, 0.36 mmol). The reactionmixture was stirred for 2 h at RT. The reaction was quenched by theaddition of water (500 μL). The mixture was concentrated andco-evaporated with toluene (3×10 mL). The residue was purified by columnchromatography (gradient PE/EtOAc 1:0 to 1:1) to afford product 14 (99mg, 0.16 mmol, 44%) as a white solid. MS (ESI) m/z calcd. forC₂₈H₅₀FN₆O₅Si₂ [M+H]⁺ 625.9; found 625.3.

Step 3. Preparation of(2R,3R,4R,5R)-5-(2-isobutylamido-6-methylamino-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(15)

To a solution of compound 14 (90 mg, 0.14 mmol) in dry THF (2 mL) wasadded tetrabutylammonium fluoride (1 M in THF, 38 μL, 0.38 mmol). Themixture was stirred for 2 h at RT and concentrated. The residue waspurified by column chromatography (gradient DCM/MeOH 10:0 to 9:1)followed by reverse phase column chromatography (gradient H₂O/MeOH 100:0to 0:100) to give product 15 (42 mg, 0.11 mmol, 77%) as a white solid.¹H NMR (300 MHz, CD₃OD) δ 8.31 (s, 1H), 6.29 (d, J=17.9 Hz, 1H),4.70-4.60 (m, 1H), 4.07-3.98 (m, 2H), 3.89 (dd, J=12.5, 3.4 Hz, 1H),3.10 (br s, 3H), 2.87 (br s, 1H), 1.23-1.16 (m, 9H). ¹⁹F NMR (282 MHz,CD₃OD) δ −163.8. MS (ESI) m/z calcd. for C₁₆H₂₄FN₆O₄ [M+H]⁺ 383.4; found383.2.

Step 4. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-isobutylamido-6-methylamino-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(16)

To a solution of compound 15 (27 mg, 0.07 mmol) in dry THF (1 mL) at 0°C. was added t-butyl magnesium chloride (1.0 M in THF, 130 μL, 0.13mmol) dropwise over 10 min. The reaction mixture was stirred 15 min at0° C. then another 15 min at room temperature. The reaction mixture wascooled down to 0° C. and a solution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate (50 mg, 0.11mmol) dissolved in dry THF (1 mL) was added dropwise over 10 min. Thereaction mixture was stirred at 0° C. for 30 min followed by 18 h atroom temperature then quenched with a saturated aq. NH₄Cl solution (2mL) and extracted with EtOAc (3×5 mL). The combined organics were driedover Na₂SO₄ and concentrated. The residue was purified by columnchromatography (gradient DCM/MeOH 100:0 to 95:5) then reverse phasecolumn chromatography (gradient H₂O/MeOH 100:0 to 0:100) to affordproduct 16 (mixture of 2 diastereoisomers, 25 mg, 0.04 mmol, 54%) as awhite solid. ¹H NMR (300 MHz, CD₃OD) δ 8.05 (s, 1H), 7.33-7.13 (m, 5H),6.27 (d, J=18.6 Hz) and 6.21 (d, J=19.1 Hz, 1H), 5.10-4.95 (m, 1H),4.93-4.78 (overlapped with H₂O, m, 1H), 4.60-4.42 (m, 2H), 4.26-4.18 (m,1H), 3.90-3.80 (m, 1H), 3.09 (br s, 3H), 2.84-2.80 (m, 1H), 1.33-1.15(m, 18H). ³¹P NMR (121 MHz, CD₃OD) δ 3.69 (s). ³¹P NMR (121 MHz, CD₃OD)δ 4.11 (s), 3.99 (s). MS (ESI) m/z calcd. for C₂H₄₀FN₇O₈P [M+H]⁺ 652.6;found 652.3.

Example 6. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-ethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(18) Step 1. Preparation of(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-ethylamino)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(17)

To a solution of compound 3 (150 mg, 0.29 mmol) in MeOH (4 mL) was addedN-methylethylamine (245 μL, 2.90 mmol). The reaction mixture was heatedat 100° C. in a sealed tube for 15 h, cooled down to room temperatureand concentrated. The residue was purified by column chromatography(gradient DCM/MeOH 100:0 to 90:10) to afford product 31 (89 mg, 0.26mmol, 89%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 8.06 (s, 1H), 6.13 (d, J=18.0 Hz, 1H), 4.40(dd, J=24.9, 8.7 Hz, 1H), 4.11-4.01 (m, 4H), 3.98-3.83 (m, 1H), 3.34(br. s, 3H), 1.24-1.11 (m, 6H). ¹⁹F NMR (282 MHz, CD₃OD) δ −163.7. MS(ESI) m/z calcd. for C₁₄H₂₂FN₆O₃ [M+H]⁺ 341.2; found 341.2.

Step 2. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-ethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(18)

To a solution of compound 17 (30 mg, 0.09 mmol) in dry THE (2 mL) at 0°C. was added tert-butyl magnesium chloride (1.0 M in THF, 110 μL, 0.11mmol) dropwise over 10 min. The reaction mixture was stirred 15 min at0° C. then another 15 min at room temperature. The reaction mixture wascooled down to 0° C. and a solution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate (48 mg, 0.11mmol) dissolved in dry THE (1 mL) was added dropwise over 10 min. Thereaction mixture was stirred at 0° C. for 30 min and 18 h at roomtemperature. The reaction was quenched with a saturated aq. NH₄Clsolution (4 mL) and extracted with EtOAc (3×5 mL). The combined organicswere dried over Na₂SO₄ and concentrated. The residue was purified bycolumn chromatography (gradient DCM/MeOH 100:0 to 90:10) to afford theproduct 18 (mixture of 2 diastereoisomers, 22 mg, 0.04 mmol, 40%) as awhite solid.

¹H NMR (300 MHz, CD₃OD) δ 7.69 (m, 1H), 7.26-7.04 (m, 5H), 6.05 (d,J=18.6 Hz) and 6.03 (d, J=18.9 Hz, 1H), 4.86-4.79 (overlapped with H₂O,m, 1H), 4.50-4.32 (m, 3H), 4.12-4.06 (m, 1H), 3.96-3.79 (m, 3H), 3.25(br. s, 3H), 1.24-1.02 (m, 15H). ³¹P NMR (121 MHz, CD₃OD) δ 4.07 (s),4.00 (s). MS (ESI) m/z calcd. for C₂₆H₃₈FN₇O₇P [M+H]⁺ 609.3; found609.2.

Example 7. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-propylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(20) Step 1. Preparation of(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-propylamino)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(19)

To a solution of compound 3 (150 mg, 0.29 mmol) in MeOH (4 mL) was addedN-methylpropylamine (295 μL, 2.90 mmol). The reaction mixture was heatedat 100° C. in a sealed tube for 15 h, cooled down to room temperatureand concentrated. The residue was purified by column chromatography(gradient DCM/MeOH 100:0 to 90:10) then reverse phase columnchromatography (gradient H₂O/MeOH 100:0 to 0:100) to afford product 19(80 mg, 0.23 mmol, 78%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 8.04 (s, 1H), 6.13 (d, J=18.3, 1H), 4.40 (dd,J=24.2, 9.2 Hz, 1H), m, 4.06-3.84 (m, 5H), 1.68 (sept, J=7.5 Hz, 2H),1.15 (d, J=22.2 Hz, 3H), 0.93 (t, J=7.5 Hz, 3H). ¹⁹F NMR (282 MHz,CD₃OD) δ −163.8. MS (ESI) m/z calcd. for C₁₅H₂₄FN₆O₃ [M+H]⁺ 355.2; found355.2.

Step 2. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-propylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(20)

To a solution of compound 19 (30 mg, 0.09 mmol) in dry THE (2 mL) at 0°C. was added tert-butyl magnesium chloride (1.0 M in THF, 110 μL, 0.11mmol) dropwise over 10 min. The reaction mixture was stirred 15 min at0° C. then another 15 min at room temperature. The reaction mixture wascooled down to 0° C. and a solution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate (46 mg, 0.11mmol) dissolved in dry THE (1 mL) was added dropwise over 10 min. Thereaction mixture was stirred at 0° C. for 30 min and 18 h at roomtemperature. The reaction was quenched with a saturated aq. NH₄Clsolution (4 mL) and extracted with EtOAc (3×5 mL). The combined organicswere dried over Na₂SO₄ and concentrated. The residue was purified bycolumn chromatography (gradient DCM/MeOH 100:0 to 90:10) to affordproduct 20 (mixture of 2 diastereoisomers, 22 mg, 0.03 mmol, 33%) as awhite solid.

¹H NMR (300 MHz, CD₃OD) δ 7.78, 7.77 (s+s, 1H), 7.37-7.13 (m, 5H), 6.15(d, J=18.6 Hz) and 6.13 (d, J=18.9 Hz, 1H), 4.97-4.89 (overlapped withH₂O, m, 1H), 4.63-4.30 (m, 3H), 4.22-4.14 (m, 1H), 4.02-3.84 (m, 2H),1.74-1.63 (3H, m), 1.32-1.27 (m, 3H), 1.23-1.13 (m, 9H), 0.94 (t, J=7.4Hz) and 0.93 (t, J=7.4 Hz, 3H). ³¹P NMR (121 MHz, CD₃OD) δ 4.05 (s),4.00 (s). MS (ESI) m/z calcd. for C₂₇H₄₀FN₇O₇P [M+H] 623.3; found 623.2.

Example 8. Preparation of Isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-cyclobutylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(22) Step 1. Preparation of(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-cyclobutylamino)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-ol(21)

To a solution of compound 3 (150 mg, 0.29 mmol) in MeOH (4 mL) was addedN-methylcyclobutylamine hydrochloride (105 mg, 0.90 mmol) andtriethylamine (190 μL, 1.00 mmol). The reaction mixture was heated at100° C. in a sealed tube for 15 h and cooled down to room temperature.An aqueous solution containing 30% NH₄OH (1 mL) was added and thereaction mixture was heated at 100° C. in a sealed tube for 2 h, cooleddown and concentrated. The residue was purified by column chromatography(gradient DCM/MeOH 100:0 to 90:10) to afford product 21 (90 mg, 0.25mmol, 86%) as a pale yellow solid.

¹H NMR (300 MHz, CD₃OD) δ 8.09 (s, 1H), 6.14 (d, J=18.0 Hz, 1H),5.80-5.70 (m, 1H), 4.44-4.33 (m, 1H), 4.06-4.02 (m, 2H), 3.88-3.84 (m,1H), 3.34 (s, 3H), 2.38-2.19 (m, 4H), 1.79-1.71 (m, 2H), 1.17 (d, J=22.2Hz, 3H). ¹⁹F NMR (282 MHz, CD₃OD) δ −163.8. MS (ESI) m/z calcd. forC₁₆H₂₄FN₆O₃ [M+H]⁺ 367.2; found 367.2.

Step 2. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methyl-cyclobutylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(22)

To a solution of compound 21 (50 mg, 0.14 mmol) in dry THE (2 mL) at 0°C. was added tert-butyl magnesium chloride (1.0 M in THF, 210 μL, 0.21mmol) dropwise over 10 min. The reaction mixture was stirred 15 min at0° C. then another 15 min at room temperature. The reaction mixture wascooled down to 0° C. and a solution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate (74 mg, 0.16mmol) dissolved in dry THE (2 mL) was added dropwise over 10 min. Thereaction mixture was stirred at 0° C. for 30 min and 18 h at roomtemperature. The reaction was quenched with a saturated aq. NH₄Clsolution (4 mL) and extracted with EtOAc (3×5 mL). The combined organicswere dried over Na₂SO₄ and concentrated. The residue was purified bycolumn chromatography (gradient DCM/MeOH 100:0 to 90:10) and then byreverse phase column chromatography (gradient H₂O/MeOH 100:0 to 0:100)to afford product 22 (mixture of 2 diastereoisomers, 24 mg, 0.04 mmol,28%) as a white solid.

¹H NMR (300 MHz, CD₃OD) δ 7.79 (s, 0.2H), 7.77 (s, 0.8H), 7.38-7.12 (m,5H), 6.18 (d, J=17.6 Hz) and 6.16 (d, J=17.5 Hz, 1H), 4.95-4.81 (m, 2H),4.62-4.43 (m, 3H), 4.25-4.18 (m, 1H), 3.96-3.83 (m, 1H), 3.38 (s) and3.36 (s, 3H), 2.38-2.21 (m, 4H), 1.75-1.63 (m, 2H), 1.32-1.16 (m, 12H).³¹P NMR (121 MHz, CD₃OD) δ 4.04 (s), 3.97 (s). MS (ESI) m/z calcd. forC₂₈H₄₀FN₇O₇P [M+H]⁺ 636.3; found 636.2.

Modification of the 2-Amino Moiety in the Active Compounds One ofordinary skill in the art can add a substituent to the 2-amino purinemoiety by methods well known to those skilled in the art. Onenon-limiting process is provided here, and others can be easily adapted.((2R,3R,4R,5R)-3-(benzoyloxy)-5-bromo-4-fluoro-4-methyltetrahydrofuran-2-yl)methylbenzoate, is treated with commercially available 2,6-dichloropurine, abase and a mixture of organic solvents at an elevated temperature togenerate(2R,3R,4R,5R)-5-(2,6-dichloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate. In one embodiment, the base is potassium tert-butoxide. In oneembodiment, the mixture of organic solvents comprises tert-butanol andacetonitrile. The compound,(2R,3R,4R,5R)-5-(2,6-dichloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate is treated with an amine, a base and an organic solvent atambient temperature to generate 2-chloro-N⁶-substituted purines. In oneembodiment, the amine is methylamine. In one embodiment, the base istriethylamine. In one embodiment, the organic solvent is ethanol. Oneskilled in the art will also recognize that upon treatment with an amineand base, the benzoate groups on the nucleoside will simultaneously beremoved to generate the deprotected furanose moiety.2-Chloro-N⁶-substituted purines can then be treated with an amine, andan organic solvent in a sealed tube at an elevated temperature of about100° C. to generate N²,N⁶-disubstituted purine nucleosides of thepresent invention. In one embodiment, the amine is methylamine. In oneembodiment, the organic solvent is ethanol. N²,N⁶-Disubstituted purinenucleosides of the present invention can be treated with a base,isopropyl ((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninateand an organic solvent at a reduced temperature to generate compounds ofFormula I-V. In one embodiment, the base is tert-butyl magnesiumchloride. In one embodiment, the organic solvent is tetrahydrofuran.

Preparation of Stereospecific Phosphorus Enantiomers

Certain of the active compounds described herein have a chiralphosphorus moiety. Any of the active compounds described herein can beprovided as an isolated phosphorus enantiomeric form, for example, atleast 80, 90, 95 or 98% of the R or S enantiomer, using methods known tothose of skill in the art. For example, there are a number ofpublications that describe how to obtain such compounds, including butnot limited to column chromatography, for example as described inExample 17 below and U.S. Pat. Nos. 8,859,756; 8,642,756 and 8,333,309to Ross, et al.

Example 9. Separation of the Stereoisomers of Compound 5

The stereoisomers of Compound 5 were separated on a Phenominex Lunacolumn using the following conditions:

Column: Phenominex Luna 5 micron C18 (2) 250×10 mm part # OOG-4252-BO

Sample concentration: Approximately 50 mg/ml in acetonitrile

Injection volume: 50

Mobile phase A: HPLC grade water

Mobile phase B: HPLC grade acetonitrile.

Flow: 5 ml/min

UV: 283 nm

Gradient:

Time % B 0 2 40 50 41 50 41.1 2 45 2

Run time: 45 minutes

Column Temperature: 40° C.

A sample chromatogram of a semi-prep run is illustrated in FIG. 1.

The combined fractions were evaluated using an analytical column withthe following conditions:

Column: Phenominex Luna 5 micron C18 (2) 250×2 mm part # OOG-4252-BO

Injection volume: 10

Mobile phase A: HPLC grade water

Mobile phase B: HPLC grade acetonitrile.

Flow: 0.2 ml/min

UV: 283 nm

Gradient:

Time % B 0 2 30 50 40 50 40.1 2 45 2

Run time: 45 minutes

Column Temperature: 40° C.

The combined fractions for each stereoisomer were evaporated to drynessusing a rotovap with a bath temperature of 30° C. The resulting solidswere dissolved in 1 ml of acetonitrile, transferred into 1.7 mlmicrocentrifuge tubes and the solvent evaporated on the vacuumcentrifuge at a temperature of 30° C.

The data on the final samples are as follows:

1. First eluding peak: Compound 5 #1 (5-1) (21.7 mgs—97.8% ee).

2. Second eluding Peak: Compound 5 #2 (5-2) (13.2 mgs—95.9% ee).

The final weights of the 1^(st) and 2^(nd) peak correspond well to theirpercentages in the original mixture. (62.2% and 37.8% respectively).

Stereospecific Syntheses of Compounds of Formula I-VII

Example 10. Preparation of(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(hydroxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ol(23) Step 1. Preparation of(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(hydroxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ol(23)

The compound(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(benzoyloxymethyl)-4-fluoro-4-methyltetrahydrofuran-3-ylbenzoate, 3, (80 g, 140 mmol) was added to a solution of trimethylaminein methanol (7 M, 800 mL) and stirred at RT overnight. The mixture wasconcentrated and then purified by column chromatography (DCM:MeOH=100:1)to afford(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(hydroxymethyl)-4-fluoro-4-methyl-tetrahydrofuran-3-ol(23) (40 g, 90%).

Example 11. Preparation of((((S)-(2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

Step 1. Preparation of(2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-ol(4)

To a solution of(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(hydroxymethyl)-4-fluoro-4-methyl-tetrahydrofuran-3-ol(2.0 g, 1.0 eq) in dioxane (15 mL) was added MeNH₂ aqueous solution (5.0eq). After stirring overnight at RT, TLC showed that the startingmaterial was consumed. The mixture was concentrated and purified bycolumn chromatography (DCM:MeOH=40:1-30:1) to afford(2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-olas a white powder (1.6 g, 81.6%). [M+H]⁺=313.5

Step 2. Preparation of((((S)-(2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

The compound(2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-ol(1.47 g, 1.0 eq) and PPAL-S (2.35 g, 1.1 eq) were dissolved in anhydrousTHE (29 mL). After cooling the mixture to −10° C., t-BuMgCl (5.8 mL, 1.7M, 2.1 eq) was slowly added under a blanket of N₂. After stirring at RTfor 45 min, the mixture was quenched with aq. saturated NH₄Cl, andextracted with EtOAc (20 mL×3). The combined organic layers were washedwith water, brine (30 mL), dried over anhydrous Na₂SO₄ and concentrated.The crude product was purified by column chromatography(DCM:MeOH=50:1-20:1) to afford((((S)-(2R,3R,4R,5R)-5-(2-amino-6-(methylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyl-tetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninateas a white powder (1.1 g, 40.3%).

¹H NMR (400 MHz, CD₃OD) δ 7.81 (s, 1H), 7.33-7.16 (m, 5H), 6.10 (d,J=18.4 Hz, 1H), 4.90-4.84 (m, 5H), 4.55-4.46 (m, 3H), 4.20-4.16 (m, 1H),3.91-3.87 (m, 1H), 3.30 (m, 1H), 3.03 (s, 3H), 1.30-1.20 (m, 12H).[M+H]⁺=582.8.

Example 12. Preparation of isopropyl((((S)-(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(25)

Step 1. Preparation of(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-ol

To a solution of(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(hydroxymethyl)-4-fluoro-4-methyl-tetrahydrofuran-3-ol(2.8 g, 8 mmol) in dioxane (20 mL) was added dimethylamine aqueoussolution (5 mL). After stirring at RT for 3 h, TLC showed that thestarting material was consumed. The mixture was concentrated andpurified by column chromatography (DCM:MeOH=60:1) to afford(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-ol(2.2 g).

¹H NMR (400 MHz, CD₃OD) δ 8.08 (s, 1H), 6.13 (d, J=18.0 Hz, 1H), 4.43(dd, J=9.2, 9.2 Hz, 1H), 4.06 (d, J=10.8 Hz, 2H), 3.90 (m, 1H), 3.37 (s,3H), 3.06 (s, 3H), 1.18 (d, J=22 Hz, 3H).

Step 2. Preparation of isopropyl((((S)-(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(25)

The compound(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-ol(8 g, 1.0 eq) and PPAL-S (11.1 g, 1 eq) were dissolved in anhydrous THF(100 mL). The mixture was cooled to −5-0° C. and t-BuMgCl (30.5 mL, 1.7M, 2.1 eq) was slowly added under a N₂ atmosphere. After stirring at RTfor 2 h, the mixture was quenched with aq. saturated NH₄Cl solution andextracted with EtOAc (70 mL×3). The combined organic layers were washedwith water, brine (30 mL), dried over anhydrous Na₂SO₄ and concentrated.The crude product was purified by column chromatography (DCM:MeOH=50:1)to afford isopropyl((((S)-(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninateas a white powder (9.5 g, 65%).

¹H NMR (400 MHz, CD₃OD) δ 7.81 (s, 1H), 7.35-7.19 (m, 5H), 6.15 (d,J=18.8 Hz, 1H), 4.90 (m, 1H), 4.54-4.49 (m, 3H), 4.22-4.19 (m, 1H), 3.90(m, 1H), 3.43 (s, 3H), 1.32 (d, J=7.2 Hz, 3H), 1.24-1.17 (m, 9H). ³¹PNMR (160 MHz, CD₃OD) δ 3.89.

Example 13. Preparation of isopropyl((((R)-(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate(26)

The compound(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-ol(3 g, 1.0 eq) and PPAL-R (4.17 g, 1 eq) were dissolved in anhydrous THE(60 mL). The mixture was cooled to −5-0° C. and t-BuMgCl (11.4 mL, 1.7M, 2.1 eq) was slowly added under a N₂ atmosphere. After stirring at RTfor 16 h, the mixture was quenched with aq. saturated NH₄Cl solution andextracted with EtOAc (50 mL×3). The combined organic layers were washedwith water, brine (30 mL), dried over anhydrous Na₂SO₄ and concentrated.The crude product was purified by column chromatography (DCM:MeOH=50:1)to afford isopropyl((((R)-(2R,3R,4R,5R)-5-(2-amino-6-(dimethylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninateas a white powder (2.2 g, 41%).

¹H NMR (400 MHz, CD₃OD) δ 7.8 (s, 1H), 7.35-7.29 (m, 5H), 6.18 (d,J=18.8 Hz, 1H), 4.92 (m, 1H), 4.60 (m, 1H), 4.51-4.23 (m, 3H), 3.90 (m,1H), 3.44 (s, 6H), 1.29 (d, J=6 Hz, 3H), 1.22-1.16 (m, 10H). ³¹P NMR(160 MHz, CD₃OD) δ 3.98.

Example 14. Preparation of isopropyl((((S)-(2R,3R,4R,5R)-5-(2-amino-6-(methylcyclopropanamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

Step 1: Preparation of(2R,3R,4R,5R)-5-(2-amino-6-(methylcyclopropanamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-ol(8)

K₂CO₃ (53 g, 500 mmol) was added to N-methylcyclopropanaminohydrochloride in aqueous solution (100 mL). After stirring at RT for 10min, a solution of(2R,3R,4R,5R)-5-(2-amino-6-chloro-9H-purin-9-yl)-2-(hydroxymethyl)-4-fluoro-4-methyl-tetrahydrofuran-3-ol(35 g, 109 mmol) in dioxane (300 mL) was added. The mixture was stirredat RT for 16 h and HPLC indicated that the reaction was complete. Themixture was concentrated and purified by column chromatography(DCM:MeOH=60:1) to afford(2R,3R,4R,5R)-5-(2-amino-6-(methylcyclopropanamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-ol(30 g, 82%).

¹H NMR (400 MHz, CD₃OD) δ 8.16 (s, 1H), 6.17 (d, J=18.0 Hz, 1H), 4.41(dd, J=9.2, 9.2 Hz, 1H), 4.06 (m, 2H), 3.90 (m, 1H), 3.37 (s, 3H), 3.16(m, 1H), 1.18 (d, J=22.4 Hz, 3H), 0.94 (m, 2H), 0.74 (m, 2H).[M+H]⁺=353.2.

Step 2: Preparation of isopropyl((((S)-(2R,3R,4R,5R)-5-(2-amino-6-(methylcyclopropanamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

The compound(2R,3R,4R,5R)-5-(2-amino-6-(methylcyclopropanamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-ol(8 g, 1.0 eq) and PPAL-S (10.3 g, 1 eq) were dissolved in anhydrous THF(100 mL). After cooling the mixture to −5-0° C., t-BuMgCl (28 mL, 1.7 M,2.1 eq) was slowly added under a N₂ atmosphere. The mixture was stirredat RT for 1 h, quenched with aq. saturated NH₄Cl solution, and extractedwith EtOAc (70 mL×3). The combined organic layers were washed withwater, brine (30 mL), dried over anhydrous Na₂SO₄ and concentrated. Thecrude product was purified by column chromatography (DCM:MeOH=100:1 to50:1) to afford isopropyl((((S)-(2R,3R,4R,5R)-5-(2-amino-6-(methylcyclopropanamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninateas a white powder (9.5 g, 65%).

¹H NMR (400 MHz, CD₃OD) δ 7.86 (s, 1H), 7.35-7.19 (m, 5H), 6.17 (d,J=19.2 Hz, 1H), 4.91 (m, 1H), 4.52 (m, 3H), 4.21 (m, 1H), 3.93 (m, 1H),3.35 (s, 3H), 3.16 (m, 1H), 2.0 (s, 1H), 1.26-1.16 (m, 12H), 0.93 (m,2H), 0.73 (m, 2H). ³¹P NMR (160 MHz, CD₃OD) δ 3.90

Example 15. Preparation of isopropyl((((R)-(2R,3R,4R,5R)-5-(2-amino-6-(methylcyclopropanamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

The compound(2R,3R,4R,5R)-5-(2-amino-6-(methylcyclopropanamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-3-ol(3 g, 1.0 eq) and PPAL-R (2.8 g, 1 eq) were dissolved in anhydrous THF(60 mL). After cooling the mixture to −5-0° C., t-BuMgCl (7.6 mL, 1.7 M,2.1 eq) was slowly added under N₂. Then the mixture was stirred at RTfor 1 h and quenched with aq. saturated NH₄Cl solution, and extractedwith EtOAc (50 mL×3). The combined organic layers were washed withwater, brine (30 mL), dried over anhydrous Na₂SO₄ and concentrated. Thecrude product was purified by column chromatography (DCM:MeOH=100:1 to50:1) to afford the product as a white powder (3 g, 55%).

¹H NMR (400 MHz, CD₃OD) δ 7.81 (s, 1H), 7.30-7.25 (m, 5H), 6.16 (d,J=24.8 Hz, 1H), 4.84 (m, 1H), 4.84-4.50 (m, 3H), 4.22-4.19 (m, 1H), 3.88(m, 1H), 3.33 (s, 3H), 3.14 (m, 1H), 2.0 (s, 1H), 1.28-1.13 (m, 12H),0.92 (m, 2H), 0.90 (m, 2H). ³¹P NMR (160 MHz, CD₃OD) δ 3.99.

Example 16. Preparation of Compound 32

Step 1. Preparation of Compound 29.

To a solution of 6 (3.0 g, 1.0 eq) in pyridine (30 mL) was addedTIPDSCl₂ (4.35 g, 1.5 eq) at 0° C. After stirring at RT for 4 h, TLCshowed that starting material was consumed. The mixture was diluted withEtOAc, washed with 1M aq. HCl solution, saturated NaHCO₃ aqueoussolution, brine, dried over anhydrous Na₂SO₄ and concentrated to afford29 as a yellow oil (6.3 g, 100%).

Step 2. Preparation of Compound 30.

To a mixture of Compound 29 (800 mg, 1.0 eq), DMAP (16 mg, 0.1 eq),pyridine (1.6 mL) and DCM (10 mL) was added isobutyryl chloride (209 mg,1.5 eq) at 0° C. After stirring at RT for 2 h, TLC showed that thestarting material was consumed. The mixture was quenched with water,washed with aq. 1M HCl solution, saturated NaHCO₃ aqueous solution,brine, dried over anhydrous Na₂SO₄ and concentrated. The crude productwas purified by column chromatography to afford the product, 30, as awhite oil (563 mg, 62.3%).

¹H NMR (400 MHz, CDCl₃) δ 7.98 (s, 1H), 787 (s, 1H), 6.20 (d, J=16.0 Hz,1H), 4.32-4.07 (m, 4H), 3.50 (s, 6H), 2.3 (m, 1H), 1.29-1.05 (m, 45H).

Step 3. Preparation of Compound 31.

To a mixture of 30 (560 mg, 1.0 eq) in THE (10 mL) was added Et₃N.3HF(706 mg, 5 eq) and Et₃N (890 mg, 10 eq) at RT. After stirring at RT for1.5 h, TLC showed that the starting material was consumed. The mixturewas concentrated and purified by column chromatography to afford 31 as awhite powder (288 mg, 83%).

¹H NMR (400 MHz, CDCl₃) δ 7.72 (s, 1H), 5.96 (d, J=44.0 Hz, 1H), 5.22(m, 1H), 4.13-3.99 (m, 4H), 3.42 (s, 6H), 2.83-2.63 (m, 2H), 1.29-1.17(m, 9H).

Step 4. Preparation of Compound 32.

Compound 31 (280 mg, 1.0 eq) and PPAL-S (320 mg, 1 eq) were dissolved inanhydrous THE (10 mL). After cooling the mixture to −5° C., t-BuMgCl(0.87 mL, 1.7 M, 2.1 eq) was slowly added under a N₂ atmosphere. Themixture was stirred at RT for 2 h, quenched with aq. saturated NH₄Clsolution, and extracted with EtOAc (10 mL×3). The combined organiclayers were washed with water, brine (20 mL), dried over anhydrousNa₂SO₄ and concentrated. The crude product was purified by columnchromatography to afford the product as a white powder (260 mg, 50%).

¹H NMR (400 MHz, CD₃OD) δ 7.98 (s, 1H), 7.25 (m, 5H), 6.23 (d, J=18.8Hz, 1H), 4.52 (m, 3H), 4.38 (m, 1H), 3.81 (m, 1H), 3.75 (m, 1H), 3.48(s, 6H), 2.81 (m, 1H), 1.32 (m, 18H). [M+H]⁺=666.9.

Example 17. Preparation of Compound 35

Step 1. Preparation of Compound 33.

To a mixture of 29 (2.0 g, 1.0 eq), DMAP (0.04 g, 0.1 eq), pyridine (4mL) and DCM (20 mL) was added AcCl (0.414 g, 1.5 eq) at 0° C. Afterstirring at RT for 2 h, TLC showed that the starting material wasconsumed. The mixture was quenched with water, washed with aq. 1M HClsolution, saturated NaHCO₃ aqueous solution then brine, dried overanhydrous Na₂SO₄ and concentrated. The crude product was purified bycolumn chromatography to afford the product, 33, as a white oil (1.73 g,80.8%).

¹H NMR (400 MHz, CDCl₃) δ 7.99 (s, 1H), 7.74 (s, 1H), 6.20 (d, J=20.0Hz, 1H), 4.33-4.11 (m, 4H), 3.50 (s, 6H), 2.63 (s, 3H), 2.3 (m, 1H),1.26-1.05 (m, 29H). [M+H]⁺=611.9.

Step 2. Preparation of Compound 34.

To a mixture of 33 (1.58 g, 1.0 eq) in THE (20 mL) was added Et₃N.3HF(2.1 g, 5 eq) and Et₃N (2.6 g, 10 eq) at RT. After stirring at RT for1.5 h, TLC showed that the starting material was consumed. The mixturewas concentrated and purified by column chromatography to afford 34 as awhite powder (782 mg, 82%). [M+H]+=369.6.

Step 3. Preparation of Compound 35.

Compound 34 (136 mg, 1.0 eq) and PPAL-S (184 mg, 1.1 eq) were dissolvedin anhydrous THE (3 mL). After cooling the mixture to −5° C., t-BuMgCl(0.5 mL, 1.7 M, 2.1 eq) was slowly added under a N₂ atmosphere. Themixture was stirred at RT for 30 min, quenched with aq. saturated NH₄Clsolution and extracted with EtOAc (10 mL×3). The combined organic layerswere washed with water, brine (20 mL), dried over anhydrous andconcentrated. The crude product was purified by column chromatography(DCM:MeOH=50:1-20:1) to afford the phosphoramidate 35 as a white powder(150 mg, 63.8%).

¹H NMR (400 MHz, CD₃OD) δ 7.81 (s, 1H), 7.35-7.16 (m, 5H), 6.10 (d,J=18.4 Hz, 1H), 4.87 (m, 1H), 4.52-4.46 (m, 3H), 4.21 (m, 1H), 3.91-3.87(m, 1H), 3.03 (s, 3H), 1.30-1.13 (m, 12H). ³¹P NMR (160 MHz, CD₃OD) δ3.84. ¹⁹F NMR (376 MHz, CD₃OD) δ −162.79.

Synthesis ofβ-D-2′-deoxy-2′-α-fluoro-2′-β-ethynyl-N⁶-substituted-2,6-diaminopurinenucleotides

Example 18. General Route toβ-D-2′-deoxy-2′-α-fluoro-2′-β-ethynyl-N⁶-substituted-2,6-diaminopurinenucleotides

Step 1. Preparation of Compound 36.

To a solution of 6-chloroguanosine (100 g, 332 mmol) in pyridine (400mL) was added TPDSCl₂ (110 mL, 1.05 eq.) dropwise at −5-5° C. under a N₂atmosphere. After stirring at that temperature for 2 h, TLC showed thestarting material was consumed. DCM (600 mL) was added, and then TMSCl(85 mL, 2 eq.) was added dropwise at 0-5° C. After stirring at thattemperature for 2 h, TLC showed the intermediate was consumed.

Isobutyryl chloride was added dropwise at 0-5° C. After stirring at thattemperature for 2 h, TLC showed the intermediate was consumed. Water wasadded, and the content was extracted with DCM. The organic phase wasthen washed with 0.5 N HCl to remove pyridine.

After thepH of the content was washed to 5˜6, pTSA.H₂O (9.2 g, 484.5mmol) was added at 0-5° C. After stirring at that temperature for 1 h,TLC showed the intermediate was consumed. Water was then added, and theorganic phase was washed with water, saturated aqueous NaHCO₃ and brine.After being dried over Na₂SO₄, the solvent was removed in vacuo. Theresidue was then purified with column chromatography (PE/EA=100-10/1) toafford the product as a light yellow solid (82 g, 40%).

¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H), 8.55 (s, 1H), 5.91 (d, J=1.6Hz, 1H), 5.53 (d, J=4.6 Hz, 1H), 4.72-4.58 (m, 2H), 4.16 (dd, J=12.4,4.8 Hz, 1H), 4.00 (ddd, J=7.7, 4.8, 2.6 Hz, 1H), 3.93 (dd, J=12.4, 2.7Hz, 1H), 2.78 (h, J=6.9 Hz, 1H), 1.26-1.12 (m, 3H), 1.10 (d, J=6.7 Hz,6H), 1.09-0.88 (m, 24H).

Step 2. Preparation of Compound 37.

To a solution of 36 (10.0 g, 16.3 mmol) in DCM (100 mL) was addedDess-Martin periodinane at rt and the reaction was stirred for 12 h. TLCshowed the starting material was consumed. The reaction mixture was thendiluted with DCM (200 mL) and washed with saturated aqueous Na₂S203 andbrine. The organic phase was then dried over Na₂SO₄ and concentrated toafford crude 37 as a light yellow solid (12 g). The crude 53 can be useddirectly in the next step without purification.

Step 3. Preparation of Compound 38.

To a solution of ethynyltrimethylsilane (18.6 mL, 142.7 mmol) in THE(240 mL) was added n-BuLi (46 mL, 2.5 M, 115.0 mmol) dropwise at−15˜−20° C. under a N₂ atmosphere. After stirring for 30 min, thereaction was cooled to −70° C., and 37 (crude, 16.3 mmol) in THE (60 mL)was added at that temperature. The content was then warmed to 0° C. TLCshowed the starting material was consumed. Saturated aqueous NH₄Cl wasadded, and the reaction was extracted with EA (100 mL) three times. Theorganic phase was combined and then washed with brine, then furtherdried over Na₂SO₄. After being concentrated in vacuo, the residue waspurified by column chromatography (PE/EA=100→10/1) to afford a lightyellow solid (6.0 g, 52%).

Step 4. Preparation of Compound 39.

To a solution of 38 (6.0 g, 8.4 mmol) in DCM (240 mL) was added pyridine(4.2 mL, 52.9 mmol) under a N₂ atmosphere. The reaction was cooled to−70° C., and DAST (12 mL, 90.4 mmol) was added. The content was thenwarmed to −30° C. TLC showed that the starting material was consumed.The reaction was poured into saturated aqueous NaHCO₃, and thenextracted with DCM (200 mL). The organic phase was washed with brine anddried over Na₂SO₄. After being concentrated in vacuo, the residue waspurified with column chromatography (PE/EA=100->10/1) to afford a lightyellow solid (3.8 g, 63%).

Step 5. Preparation of Compound 40.

To a solution of 39 (3.8 g, 5.3 mmol) in THE (120 mL) was added AcOH(1.3 g, 22 mmol) and TBAF (4.2 g, 15.9 mmol) at rt. The reaction wasstirred at rt for 30 min. TLC showed the starting material was consumed.After being concentrated in vacuo, the residue was purified with columnchromatography (EA) to afford the product as a white solid (2.0 g, 95%).

General Procedure for Amino Displacement and Deprotection:

To a solution of 40 (350 mg, 0.88 mmol) in dioxane (20 mL) was added themethanol or water solution of the corresponding amine (free base or saltas hydrochloride plus DIEA) at rt. The content was stirred at rt for1-12 h. TLC showed the starting material was consumed. After beingconcentrated in vacuo, the residue was used directly in the next stepwithout purification. The above mentioned residue was dissolved inmethanol (10 mL). Aqueous NaOH (2.5 N, 10 mL) was added. After stirringovernight at rt, TLC showed that starting material was consumed. The pHof the content was adjusted to 7-8 with 1 N HC. The solution wasconcentrated and purified with column chromatography(DCM/MeOH=100->20/1) to afford the product as an off-white solid (yield:40-80% over two steps). Table 1 illustrates the structures of compounds57-63 and the corresponding mass spectral and ¹H NMR for the respectivecompounds.

TABLE 1 Compound No. Structure ¹H NMR/MS 41

¹H NMR (400 MHz, Methanol-d₄) δ 8.05 (s, 1H), 6.27 (d, J = 16.9 Hz, 1H),4.75 (dd, J = 21.7, 9.1 Hz, 1H), 4.06 (dd, J = 11.0, 2.4 Hz, 2H), 3.87(dd, J = 13.1, 3.2 Hz, 1H), 3.42 (s, 6H), 3.37 (s, 2H), 3.18 (d, J = 5.4Hz, 1H). [M + H]⁺ = 336.9 42

¹H NMR (400 MHz, DMSO-d₆) δ 7.94 (s, 1H), 7.30 (s, 1H), 6.20-6.09 (m,2H), 5.98 (s, 2H), 5.33 (t, J = 5.3 Hz, 1H), 4.57 (dt, J = 22.1, 8.0 Hz,1H), 4.12 (q, J = 5.3 Hz, 1H), 3.91 (d, J = 9.3 Hz, 1H), 3.70 (t, J =8.6 Hz, 1H), 3.36 (s, 1H), 3.18 (d, J = 5.2 Hz, 2H), 2.89 (d, J = 7.0Hz, 3H). [M + H]⁺ = 323.0 43

¹H NMR (400 MHz, Methanol-d₄) δ 8.11 (s, 1H), 6.29 (d, J = 16.9 Hz, 1H),4.76 (dd, J = 21.7, 9.0 Hz, 1H), 4.10-4.01 (m, 2H), 3.87 (dd, J = 13.1,3.1 Hz, 1H), 3.37 (s, 1H), 3.24-3.11 (m, 2H), 1.00-0.87 (m, 2H), 0.74(td, J = 4.6, 2.8 Hz, 2H). [M + H]⁺ = 363.0 44

¹H NMR (400 MHz, Methanol-d₄) δ 8.07 (s, 1H), 6.26 (d, J = 16.9 Hz, 1H),4.76 (dd, J = 21.8, 9.3 Hz, 1H), 4.11-4.01 (m, 2H), 3.89 (d, J = 3.0 Hz,1H), 3.89-3.75 (m, 1H), 3.37 (s, 2H), 3.21 (d, J = 5.4 Hz, 1H),2.97-2.86 (m, 1H), 1.00- 0.77 (m, 2H), 0.67-0.46 (m, 2H). [M + H]⁺=348.8

Example 19. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-dimethylamino-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-ethynyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

Step 1. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-dimethylamino-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-ethynyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

To a solution of compound 41 (30 mg, 0.09 mmol) in dry THE (2 mL) at 0°C. was added tert-butyl magnesium chloride (1.0 M in THF, 125 μL, 0.13mmol) dropwise over 10 min. The reaction mixture was stirred 15 min at0° C. then another 15 min at room temperature. The reaction mixture wascooled down to 0° C. and a solution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate (49 mg, 0.11mmol) dissolved in dry THE (2 mL) was added dropwise over 10 min. Thereaction mixture was stirred at 0° C. for 30 min and 18 h at roomtemperature. The reaction was quenched with a saturated aq. NH₄Clsolution (4 mL) and extracted with EtOAc (3×5 mL). The combined organicswere dried over Na₂SO₄ and concentrated. The residue was purified bycolumn chromatography (gradient DCM/MeOH 100:0 to 90:10) to afford theproduct (mixture of 2 diastereoisomers, 12 mg, 0.02 mmol, 24%) as awhite solid.

¹H NMR (300 MHz, CD₃OD) δ 7.79 (s, 0.45H), 7.77 (s, 0.55H), 7.36-7.14(m, 5H), 6.28 (d, J=17.4 Hz) and 6.26 (d, J=17.5 Hz, 1H), 5.00-4.44 (m,5H), 4.23-4.16 (m, 1H), 3.69-3.81 (m, 1H), 3.42 (bs, 3H), 3.40 (bs, 3H),1.32-1.26 (m, 3H), 1.20-1.15 (m, 6H). ³¹P NMR (121 MHz, CD₃OD) δ 4.04(s), 3.98 (s). MS (ESI) m/z calcd. for C₂₆H₃₄FN₇O₇P [M+H]⁺ 606.2; found606.2.

Example 20. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-methylamino-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-ethynyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

Step 1. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-methylamino-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-ethynyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

To a solution of compound 42 (30 mg, 0.09 mmol) in dry THE (2 mL) at 0°C. was added tert-butyl magnesium chloride (1.0 M in THF, 125 μL, 0.13mmol) dropwise over 10 min. The reaction mixture was stirred 15 min at0° C. then another 15 min at room temperature. The reaction mixture wascooled down to 0° C. and a solution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate (49 mg, 0.11mmol) dissolved in dry THE (2 mL) was added dropwise over 10 min. Thereaction mixture was stirred at 0° C. for 30 min and 18 h at roomtemperature. The reaction was quenched with a saturated aq. NH₄Clsolution (4 mL) and extracted with EtOAc (3×5 mL). The combined organicswere dried over Na₂SO₄ and concentrated. The residue was purified bycolumn chromatography (gradient DCM/MeOH 100:0 to 90:10) to afford theproduct (mixture of 2 diastereoisomers, 9 mg, 0.02 mmol, 18%) as a whitesolid.

¹H NMR (300 MHz, CD₃OD) δ 7.81, 7.79 (0.9 s+0.1 s, 1H), 7.36-7.14 (m,5H), 6.26 (d, J=17.4 Hz, 0.1H) and 6.24 (d, J=17.4 Hz, 0.9H), 4.93-4.89(overlapped with H₂O, m, 1H), 4.80-4.78 (m, 1H), 4.53-4.49 (m, 2H),4.21-4.18 (m, 1H), 3.95-3.84 (m, 1H), 3.23-3.20 (m, 1H), 3.04 (bs, 1H),1.31-1.14 (m, 9H). ³¹P NMR (121 MHz, CD₃OD) δ 4.06 (s), 3.97 (s). MS(ESI) m/z calcd. for C₂₅H₃₂FN₇O₇P [M+H]⁺ 592.2; found 592.2.

Example 21. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methylcyclopropylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-ethynyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

Step 1. Preparation of isopropyl((((R,S)-(2R,3R,4R,5R)-5-(2-amino-6-(N-methylcyclopropylamino)-9H-purin-9-yl)-4-fluoro-3-hydroxy-4-ethynyltetrahydrofuran-2-yl)methoxy)-phenoxy-phosphoryl)-L-alaninate

To a solution of compound 43 (40 mg, 0.11 mmol) in dry THE (2 mL) at 0°C. was added tert-butyl magnesium chloride (1.0 M in THF, 160 μL, 0.16mmol) dropwise over 10 min. The reaction mixture was stirred 15 min at0° C. then another 15 min at room temperature. The reaction mixture wascooled down to 0° C. and a solution of isopropyl((R,S)-(pentafluorophenoxy)-phenoxy-phosphoryl)-L-alaninate (55 mg, 0.12mmol) dissolved in dry THE (2 mL) was added dropwise over 10 min. Thereaction mixture was stirred at 0° C. for 30 min and 18 h at roomtemperature. The reaction was quenched with a saturated aq. NH₄Clsolution (4 mL) and extracted with EtOAc (3×5 mL). The combined organicswere dried over Na₂SO₄ and concentrated. The residue was purified bycolumn chromatography (gradient DCM/MeOH 100:0 to 90:10) to afford theproduct (mixture of 2 diastereoisomers, 18 mg, 0.03 mmol, 26%) as awhite solid.

¹H NMR (300 MHz, CD₃OD) δ 7.84, 7.82 (s+s, 1H), 7.35-7.14 (m, 5H), 6.30(d, J=17.4 Hz) and 6.26 (d, J=17.6 Hz, 1H), 4.99-4.89 (overlapped withH₂O, m, 1H), 4.82-4.69 (m, 1H), 4.59-4.46 (m, 2H), 4.21 (m, 1H),3.96-3.82 (m, 1H), 3.24-3.22 (m, 1H), 3.17-3.11 (m, 1H) 1.31-1.26 (m,3H), 1.20-1.15 (m, 6H), 0.93-0.89 (m, 2H), 0.75-0.68 (m, 2H). ³¹P NMR(121 MHz, CD₃OD) δ 4.06 (s), 3.98 (s). MS (ESI) m/z calcd. forC₂₈H₃₆FN₇O₇P [M+H]⁺ 632.2; found 632.2.

Example 22. Preparation of PPAL-S

Step 1. Preparation of Racemic PPAL

To a stirred solution of phenyl dichlorophosphate (250 g) in EtOAc (800mL) was added isopropyl L-alaninate (200 g) in triethylamine (120 g) at−10° C. The reaction was stirred at −10° C. for 1 h. The compound2,3,4,5,6-pentafluorophenol (220 g) in triethylamine (120 g) and EtOAc(400 mL) was added at −5° C. and stirred at that temperature for 0.5 h.The reaction mixture was allowed to warm to 25° C. and stirred at thattemperature for 2 h. The solution was filtrated and washed with EtOAc(2×200 mL), and the combined organic phases were evaporated under vacuumto afford the solid PPAL-RS (racemate).

Step 2. Preparation of PPAL-RS

To a stirred solution of PPAL-RS in EtOAc (200 mL) and n-heptane (1.4 L)was added 2,3,4,5,6-pentafluorophenol (10.1 g) in triethylamine (6 g),and stirring was continued for about 4-8 h. After the R-isomer of thesolid was less than 0.5%, the solid was filtered. The solid wasdissolved in EtOAc (4 L), washed with water (2×100 mL), brine (1 L),dried over anhydrous Na₂SO₄, and filtered. The solvent was removed undervacuum to afford the PPAL-S (350 g). ¹H NMR (400 MHz, DMSO-d6)δ=7.42-7.40 (m, 2H), 7.24-7.22 (m, 3H), 6.87 (dd, J=14.1, 9.9 Hz, 1H),4.90-4.84 (m, 1H), 3.94-3.88 (m, 1H), 1.27 (dd, J=7.1, 1.1 Hz, 3H), 1.15(dd, J=6.2, 1.2 Hz, 6H) ppm. ¹³P NMR (160 MHz, DMSO-d6) δ=0.37 ppm.

Example 23. Preparation of PPAL-R

To a three-necked round bottom flask fitted with a mechanic stirrer wereadded phenyl dichlorophosphate (1896 g, 0.90 mol) and anhydrous EtOAc(750 mL). The solution was cooled to −10° C. under a nitrogenatmosphere. Iso-propyl L-alaninate (118 g, 0.90 mmol) and triethylamine(100 g, 1.1 eq) were added to the above solution. A pre-cooled (below10° C.) mixture of 2,3,4,5,6-pentafluorophenol (165 g, 1 eq) andtriethylamine (90.5 g, 1 eq) in EtOAc (300 mL) was added to the mixturevia an addition funnel at −5° C. and the resulting mixture was stirredbetween 20-25° C. for 1 hour. The white precipitate (TEA.HCl) wasfiltered off and rinsed with EtOAc. The filtrate was concentrated underreduced pressure to yield PPAL-RS about 280 g (S/R=1/1) as a white solidPPAL-RS (280 g) was triturated in 300 mL of heptane/EtOAc (20:1) at roomtemperature for 5 min. The white suspension was filtered and the solidwas rinsed with a mixture of heptane/EtOAc (20:1). The filtrate wascooled to 8° C. and the solid was collected by filtration, Crude PPAL-R(10 g) was obtained with 95% chiral purity. The crude product waspurified following above step. PPAL-R (5 g) was obtained in NLT 98%chiral purity,

¹H NMR (400 MHz, DMSO-d₆) δ=7.43-7.39 (m, 2H), 7.27-7.22 (m, 3H), 6.87(dd, J=14.1, 9.9 Hz, 1H), 4.89-4.85 (m, 1H), 3.95-3.90 (m, 1H), 1.27(dd, J=7.1, 1.1 Hz, 3H), 1.14 (dd, J=6.2, 1.2 Hz, 6H). ¹³P NMR (160 MHz,DMSO-d₆) δ=0.35.

Example 24: Preparation of Compound 52

Step 1. Preparation of Compound 49.

To a solution of 48 (1.81 g, 3.23 mmol) in dioxane (18 mL) was added 40aqueous CH₃NH₂ solution (16.2 mmol). The reaction was stirred at 40° C.for 2 h. The mixture was concentrated, diluted with EtOAc (50 mL),washed with water and brine. The organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated to afford a white solid 49 (1.66 g,92).

Step 2. Preparation of Compound 50.

To a solution of 49(1.34 g, 2.42 mmol) and 1-methylimidazole (794 mg,9.68 mmol) in DCM (14 mL) was slowly added pentyl chloroformate (547 mg,3.63 mmol) at 0° C. The reaction was stirred at r.t overnight. Themixture was concentrated, and purified by column chromatography(PE:EtOAc=5:1-2:1) to afford 50 (1.01 g, 62%) as a white solid. ¹HNMR(400 MHz, DMSO) δ 7.96 (s, 1H), 6.73 (s, 1H), 6.06-6.10 (d, J=16.0 Hz,1H), 4.09-4.30 (m, 2H), 3.97-4.09 (m, 4H), 3.28 (s, 3H), 1.39-1.46 (m,2H), 1.0-1.2 (m, 35H), 0.73-0.76 (t, J=8.0 Hz, 3H).

Step 3. Preparation of Compound 51.

To a solution of 50 (1.00 g, 1.5 mmol) in THE (11 mL) was added Et₃N(2.0 mL, 15 mmol) and Et₃N.3HF (1.21 g, 7.5 mmol) at 0° C. The reactionwas stirred at r.t for 1.5 h. The mixture was concentrated, and purifiedby column chromatography (MeOH:CH₂Cl₂=50:1) to afford 75 (460 mg, 72.2%)as a white powder.

Step 4. Preparation of Compound 52.

To a solution of 51 (460 mg, 1.08 mmol) and PPAL-S (538 mg, 1.19 mmol)in anhydrous THE (9 mL) was slowly added t-BuMgCl (2.27 mmol) at 5-10°C. under N₂. The reaction was stirred at r.t for 40 min. The mixture wasquenched with aq. saturated NH₄Cl solution, extracted with EtOAc, washedwith aq. 5% K₂CO₃ solution and brine, dried over anhydrous Na₂SO₄,filtered and concentrated. The crude product was purified by columnchromatography (CH₂C₂:MeOH=15:1) to afford 52 (280 mg, 37.3%) as a whitesolid.

¹H NMR (400 MHz, DMSO) δ 8.12 (s, 1H), 7.34-7.38 (m, 2H), 7.18-7.23 (m,3H), 6.74 (s, 2H), 6.11-6.16 (d, J=16.0 Hz, 1H), 5.99-6.05 (m, 1H), 5.84(m, 1H), 4.77-4.81 (m, 1H), 4.30-4.41 (m, 3H), 4.03-4.11 (m, 3H),3.78-3.80 (m, 1H), 3.3 (s, 3H), 1.44-1.51 (m, 2H), 1.00-1.21 (m, 16H),0.76-0.80 (t, J=8.0 Hz, 3H). [M+H]⁺=696.6.

Example 25: Preparation of Compound 56

Step 1. Preparation of Compound 48.

To a solution of 23 (600 mg, 1 eq) in pyridine (30 mL) was addedTIPDSCl₂ (1.5 eq) at 0° C. The resulting solution was allowed to standat room temperature for 2 h. The mixture was quenched with ice water andextracted with EtOAc. The organic layer was washed with 1M aq. HClsolution, saturated aqueous sodium bicarbonate and saturated aqueoussodium chloride, dried over anhydrous sodium sulfate, and concentratedto yield the crude residue. The residue was purified by chromatography(MeOH:CH₂Cl₂=1:50) to afford 48 (998 mg, 94.4%) as a white solid foam.

Step 2. Preparation of Compound 53.

A mixture of 48 (800 mg, 1 eq), pyridine (3.2 mL), DMAP (34.9 mg, 0.2eq) in DCM (20 mL) was stirred at room temperature. N-amyl chloroformate(3.2 mL) was added dropwise at 0° C., and the mixture was stirred atroom temperature for 1 day. The organic layer was washed with 1M aqueousHCl solution, saturated aqueous sodium bicarbonate and saturated aqueoussodium chloride, dried over anhydrous sodium sulfate, and evaporated invacuo. The residue was purified by chromatography on silica gel(MeOH:CH₂Cl₂=1:50) to afford 53 (255 mg, 26%) as a white solid foam.

Step 3. Preparation of Compound 54.

To the solution of 53 (270 mg, 1 eq) in 1,4-dioxane (10 mL), wasdropwise added 40% aqueous CH₃NH₂ solution (225.7 mg, 5 eq). The mixturewas stirred for 2 h at room temperature and then concentrated in vacuo.The residue was chromatographed on silica gel(methanol:dichloromethane=1:40) to afford 54 (220 mg, 81.7%) as a whitesolid foam.

Step 4. Preparation of Compound 55.

Triethylamine (1011.9 mg, 10 eq) and Et₃N.3HF (806.05 mg, 5 eq) wereadded to an ice-cooled solution of 54 (668 mg, 1 eq) in THE (10 mL), themixture was stirred for 2 h at room temperature. The mixture wasconcentrated and chromatographed on silica gel (MeOH:CH₂C₂=1:30) toafford 55 (492 mg, 84%) as a white solid foam.

Step 5. Preparation of Compound 56.

To the mixture of 55 (113 mg, 1 eq) and PPAL-S (120 mg, 1 eq) in THE (4mL) was dropwise added 1.7 M t-BuMgCl in THE (0.327 mL, 2.1 eq) at −10°C. The mixture was stirred at room temperature for 1 h, and thenquenched with saturated aq. NH₄Cl solution. The aqueous phase wasextracted with EtOAc and the organic phase was washed with brine, driedand concentrated to obtain crude residue. The residue was subjected toflash chromatography to afford 56 (126 mg, 68.5%) as a white solid.

¹H NMR (400 MHz, DMSO) δ 8.00 (s, 1H), 7.10-7.45 (m, 5H), 6.15-6.20 (d,J=20.0 Hz, 1H), 5.00-5.25 (s, 1H), 4.80-4.86 (m, 1H), 4.45-4.70 (m, 2H),4.12-4.19 (m, 3H), 3.80-3.85 (m, 1H), 3.04 (s, 3H), 1.60-1.75 (m, 2H),1.10-1.40 (m, 16H), 0.76-0.80 (t, J=8.0 Hz, 3H).

³¹P NMR (160 MHz, DMSO) δ 3.57. [M+H]⁺=696.5.

Example 26: Preparation of Compound 60

Step 1. Preparation of Compound 57.

To a solution of 6 (20 g, 1 eq) in CH₃CN (100 mL) was added imidazole(16.6 g), TIPDSCl₂ (28.9 g, 1.5 eq) in sequence at 5±5° C. The resultingsolution was allowed to stand at room temperature for 4 h. The mixturewas quenched with ice water and extracted with EtOAc. The organic layerwas washed with water, saturated aqueous sodium bicarbonate andsaturated aqueous sodium chloride, dried over anhydrous sodium sulfate,and concentrated to afford the crude residue (32 g).

Step 2. Preparation of Compound 58.

To the solution of 57 (9.8 g, 1 eq) in TH (4 mL) was dropwise added 1.7M t-BuMgCl in THE (50 mL, 4.8 eq) at 0-5° C. The mixture was stirred atroom temperature for 0.5 h, and n-amyl chloroformate (2.7 g, 1.05 eq)was slowly added. The mixture was stirred at 0-5° C. for 3-4 h. Themixture was quenched with saturated aq. NH₄Cl solution. The aqueousphase was extracted with EtOAc (200 mL) and the organic phase was washedwith brine, dried and concentrated to obtain 58 (10.7 g) as oil.

Step 3. Preparation of Compound 59.

Triethylamine (10.119 g) and Et₃N.3HF (8.6 g, 5 eq) were added to anice-cooled solution of 58 (7.3 g, 1 eq) in THE (100 mL) and the mixturewas stirred for 1 h at room temperature. The mixture was concentratedand chromatographed on silica gel (MeOH:CH₂Cl₂=1:30) to afford 59 (4.3g, 91%) as a white solid.

Step 4. Preparation of Compound 60.

To the mixture of 59 (2 g, 1 eq) and PPAL-S (2.3 g, 1.1 eq) in TH (40mL) was dropwise added 1.7 M t-BuMgCl in THE (5.6 mL, 2.1 eq) at −5° C.The mixture was stirred at −20±5° C. for 1 h, and then quenched withsaturated aq. NH₄Cl solution. The aqueous phase was extracted with EtOAcand the organic phase was washed with brine, dried and concentrated toobtain crude residue. The residue was subjected to flash chromatographyto afford 60 (1.5 g, 47%) as a white solid.

¹H NMR (400 MHz, CD₃OD) δ 7.9 (s, 1H), 7.1˜7.2 (m, 5H), 6.2 (d, J=20 Hz,1H), 5.1 (br, 1H), 4.84 (m, 1H), 4.49 (m, 2H), 4.16 (m, 1H), 4.13 (m,2H), 3.86 (m, 1H), 3.45 (br, 6H), 1.70 (m, 2H), 1.26 (m, 4H), 1.20 (m,6H), 1.14 (m, 6H), 0.93 (m, 3H). [M+H]⁺=710.5.

Biological Data Example 27. Assay Methodology and Additional BiologicalData

Huh-7 luc/neo ET cells bearing a discistronic HCV genotype 1b luciferasereporter replicon were plated at 7.5×10³ cells/ml in duplicate 96-wellplates for the parallel determination of antiviral efficacy (EC₅₀) andcytotoxicity (TC₅₀). The plates were cultured for 24 hours prior to theaddition of compounds. Six serial one half log dilutions of the testarticles (high test concentration of 100.0 μM or high test concentrationof 1.0 M) and human interferon-alpha2b (high test 10.0 U/ml) wereprepared in cell culture medium and added to the cultured cells intriplicate wells for each dilution. Six wells in the test platesreceived medium alone as an untreated control. Following 72 hours ofculture in the presence of compound, one of the plates was used for thedetermination of cytotoxicity by staining with XTT and the other forantiviral efficacy by determination of luciferase reporter activity.Cytotoxicity and efficacy data were collected and imported into acustomized Excel workbook for determination of the TC₅₀ and EC₅₀ values.Data for compounds of Formula I-VII are illustrated in Table 7 below. Inaddition, FIG. 2 illustrates the HCV replication inhibition curves forCompound 5-2 and Sofosbuvir. As can be seen in FIG. 2, Compound 5-2 hasan EC₅₀=4 nM, while Sofosbuvir has an EC₅₀=53 nM. The y-axis is thepercent of virus control and the x-axis is the concentration of drug inμM. FIG. 3 illustrates the HCV replication inhibition curves forCompound 25 and Sofosbuvir. Compound 25 has an EC₅₀=4 nM and Sofosbuvirhas an EC₅₀=53 nM. The y-axis is the percent of virus control and thex-axis is the concentration of drug in M. FIG. 4 illustrates anintra-assay comparison of the anti-HCV activity for Compounds 5-2, 25,27 and Sofosbuvir. The y-axis is the percent of virus control and thex-axis is the concentration of drug in M.

Various patient-derived HCV genotypes containing wild-type andresistance-associated variants were used to determine their relativereplication sensitivity to test compounds. Replicon resistance testvectors (RTVs) containing the NS5B genomic regions were prepared usingviral RNA isolated from plasma of HCV patients. Each NS5B region wasamplified by reverse-transcription polymerase chain reaction and clonedinto an HCV replicon RTV which was then transferred by electroporationinto Huh-7 cells. After incubation in the absence and presence ofserially diluted test compounds for 72-96 hr, viral replication wasmeasured by luciferase activity and 50% inhibitory concentrations (IC₅₀values) were determined.

Table reports the IC₅₀ and IC₉₅ values for compound 25, 27, 5-2 andSofosbuvir against various clinical isolates containing wild-type andresistance-associated variants.

All compounds were significantly more effective against HCV replicationthan sofosbuvir and neither 25, 27, nor 5-2 compound showed any evidenceof cross-resistance to L159F, L159F and S282T, and C316N mutants.

TABLE 2 Antiviral Activity of Test Compounds in Patient-derived HCVGenotypes Fold Fold HCV NS5B Test IC₅₀ IC₉₅ Change in Change in Geno-Mu- Com- Value Value IC₅₀ from IC₉₅ from type tation pound (nM) (nM)Sofosbuvir Sofosbuvir 1a none sofosbuvir 62.7 507.7 25 4.4 31.3 14.216.2 27 4.2 26.4 15.0 19.3 5-2 10.5 60.8 6.0 8.4 1b none sofosbuvir 86.0642.2 1.0 25 5.9 32.0 1.0 20.0 27 5.0 28.9 0.9 22.2 5-2 10.6 72.4 0.88.9 2a none sofosbuvir 22.5 195.1 25 2.7 22.2 8.4 8.8 27 2.9 16.2 7.912.0 5-2 6.2 45.4 3.6 4.3 2b none sofosbuvir 44.8 295.3 25 3.0 14.9 15.219.9 27 3.1 14.7 14.4 20.1 5-2 6.3 32.5 7.1 9.1 3a-1 none sofosbuvir125.9 689.8 25 5.1 27.8 24.5 24.8 27 4.4 25.4 28.4 27.2 5-2 11.8 59.310.7 11.6 3a-2 none sofosbuvir 123.5 808.1 25 4.7 24.2 26.3 33.4 27 4.523.3 27.5 34.6 5-2 10.4 56.5 11.9 14.3 4a none sofosbuvir 74.9 681.4 254.6 33.0 16.2 20.7 27 3.6 38.1 20.7 17.9 5-2 9.9 74.4 7.5 9.2 4d nonesofosbuvir 93.7 1019.7 25 5.9 44.2 16.0 23.1 27 5.6 38.4 16.7 26.6 5-214.0 79.9 6.7 12.8 1a L159F sofosbuvir 114.7 1067.5 25 5.2 40.4 22.026.4 27 5.1 36.2 22.3 29.5 5-2 13.0 95.3 8.8 11.2 1a L159F sofosbuvir1619.9 16950.9 and 25 17.2 158.5 94.0 107.0 S282T 27 14.9 141.6 108.4119.7 5-2 38.7 313.5 41.9 54.1 1b C316N sofosbuvir 73.9 472.8 25 3.218.1 23.1 26.2 27 3.1 16.5 23.5 28.7 5-2 7.7 42.7 9.6 11.1

A transient transfection assay was performed to determine thesensitivity of the wild type S282T mutant of HCV to test compounds.Huh-7 cells were electroporated in the presence of RNA transcribed fromwild type or S282T HCV replicon plasmids from the T7 promoter. Thetransfected cells were seeded in to 96-well plates at 7.5×10³ cells perwell in Dulbecco's Modified Eagle's medium. After 24 hr of incubation,medium was removed and replaced with fresh medium containing no orvarious concentrations of test compounds. Following an additional 96-hrincubation, the anti-HCV activity was measured by luciferase endpointwith Britelite™ Plus luminescence reporter gene kit (Perkin Elmer,Shelton, Conn.). Duplicate plates were treated and incubated in parallelfor assessment of cellular toxicity by staining with the tetrazolium dyeXTT.

Table 3 reports the IC₅₀ and IC₉₅ values for compounds 25, 27, 5-2 andSofosbuvir against HCV wild type and S282T replicons.

All compounds were significantly more effective against HCV replicationthan sofosbuvir and neither 25, 27, nor 5-2 compounds showed anyevidence of cross-resistance to S282T variant.

TABLE 3 Antiviral Activity of Test Compounds in a HCV TransientInfection Assay Fold Fold IC₅₀ IC₉₅ change in change in NS5B Value valueIC₅₀ from IC₉₅ from Compound Mutation (nM) (nM) Sofosbuvir Sofosbuvir5-2 None 1.4 9.98 26 22.2 S282T 2.8 20.6 99.3 >48.5 25 None <1 2.7 >36.480.7 S282T <1 9.4 >278 >106.4 27 None <1 4.1 >36.4 53.2 S282T <111.8 >278 >84.7 Sofosbuvir None 36.4 218 S282T 278 >1000

The stability of selected compounds in fresh human whole blood and inhuman liver S9 fraction was determined in incubations containing 10 μMtest compound. After incubations of 0, 30, 60 min, and up to 120 min,aliquots were removed and immediately extracted with 3 volumes ofice-cold methanol/acetonitrile (1:1, v/v). Extracts were centrifuged andsupernatants were analyzed by LC-MS/MS for concentrations of unchangedtest compound and potential metabolites.

FIG. 5 illustrates the excellent stability of compound 5-2 and all2-amino derivatives in human blood.

Interestingly, FIG. 6 illustrates the in vitro time course dealkylationof the2′-deoxy-2′-α-fluoro-2′-β-methyl-N²-methyl-N⁶-methyl-2,6-diaminopurinenucleoside phosphoramidate to2′-deoxy-2′-α-fluoro-2′-β-methyl-N⁶-methyl-2,6-diaminopurine nucleosidephosphoramidate with a human liver S9 fraction. Furthermore, unexpected,faster, and a more extensive rate of cleavage of the carbamate moiety byhuman liver S9 fraction was observed as compared to compound 5-2 and itsother 2-amino derivatives (FIG. 7).

Example 28. HCV (gt1b) NS5B Polymerase Assay

Inhibition of HCV (gt1b) NS5B polymerase was determined in triplicate bymeasuring de novo polymerization in reaction mixtures containing serialdilutions of TA, in vitro transcribed viral RNA complementary to the HCV(−) strand 3′UTR region, polymerase, radiolabeled ribonucleotide, 250 μMnon-competing rNTPs, and 1 μM competing rNTP. TA concentrations thatproduced 50% inhibition (IC₅₀) were determined from resulting inhibitioncurves.

Example 29. Human Bone Marrow Progenitor Cell Assay

Fresh human bone marrow progenitor cells (Invitrogen) suspended ineither BFU-E or GM-CSF-specific culture medium were added, at 10⁵cells/well, to triplicate serial dilutions of TA in 6-well plates. After14-day incubations, colony counts were used to determine CC₅₀ values.BFU-E colonies were confirmed using the benzidene technique.

Compounds 25, 27 and 5-2 show no cytotoxicity against bone marrow stemcells in vitro.

Example 30. iPS Cardiomyocyte Assay

iPS Cardiomyocytes (Cellular Dynamics) were seeded in microliter platesat 1.5×10⁴ cells per well. After 48-hr incubation, cells were washed andmaintenance medium containing serially diluted TA was added intriplicate. After incubating for an additional 3 days, cell viabilitywas measured by staining with XTT and CC₅₀ values were calculated.

Compounds 25, 27 and 5-2 show no cytotoxicity against iPS cardiomyocytesin vitro.

Example 31. Human DNA Polymerase Assays

Inhibition of human DNA polymerases α, β and γ (CHIMERx) was determinedin triplicate in reaction mixtures of serially diluted TA, 0.05 mM dCTP,dTTP, and dATP, 10 μCi [³²P]-α-dGTP (800 Ci/mmol), 20 μg activated calfthymus DNA and additional reagents specific for each polymerase. After30-min incubations, incorporation of [α-³²P]-GTP was measured andresulting incubation curves were used to calculate IC₅₀ values.

The triphosphate, β-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-guaninetriphosphate, as well as the triphosphate analogs of compounds 25, 27and 5-2 do not inhibit human DNA polymerases α, β or γ.

Example 32. Human Hepatocyte Co-Cultures

Cytotoxicity and hepatocyte health were assessed in triplicate bymeasuring ALT leakage, urea production, albumin secretion and cellularATP contents in micro-patterned human hepatocyte co-cultures(HepatoPac®, Hepregen Corporation) prepared by seeding cryopreservedfemale human hepatocytes (single donor) and 3T3 J2 mouse fibroblasts inmicrotiter plates according to procedures established by Hepregen.Culture media was replaced with fresh media containing TA, test article,(0, 1, 10 or 30 μM) every 2 or 3 days through day 16. Spent culturemedia was assayed for ALT and urea content on days 2, 5, 7, 9, 12, 16and 21 and for albumin content on days 2, 5, 7 and 9. Cellular ATPlevels were measured on days 9 and 21. ATP signals in stromal-onlycontrol cultures (murine 3T3 fibroblasts) were subtracted from those ofhuman HepatoPac co-cultures to obtain hepatocyte-specific effects. See,Table 4, 5 and 6 below.

Compound 5-2 at concentrations up to 30 μM, showed no signs ofcytotoxicity as measured by ALT leakage, albumin secretion, ureaproduction and cellular ATP content when incubated for up to 12 dayswith micro-patterned co-cultured human hepatocytes. The minorindications of cytotoxicity detected with extended exposure (up to 21days of culture) were significantly less than those observed withsofosbuvir. See, Table 4, 5 and 6 below.

INX-189 was highly cytotoxic to human co-cultured hepatocytes, showingdecreased albumin secretion as early as day 2 and cytotoxicity by allmeasures. Sofosbuvir showed more cytotoxicity than AT-511 under the sameconditions.

TABLE 4 Effect of Test Article on Cellular ATP Concentrations 50%Inhibitory Concentration (IC₅₀) - μM Test Article Day 9 Day 21 Cmpd5-2 >30 12.8 Sofosbuvir 8.6 2.3 INX-189 8.1 0.1

TABLE 5 Effect of Test Articles on Albumin Secretion 50% InhibitoryConcentration (IC₅₀) - μM Test Article Day 2 Day 5 Day 7 Day 9 Cmpd5-2 >30 >30 >30 >30 Sofosbuvir >30 19.5 10.9 9.3 INX-189 13.6 3.1 3.22.4

TABLE 6 Effect of Test Articles on Albumin Secretion Test 50% InhibitoryConcentration (IC₅₀)-μM Article Day 2 Day 5 Day 7 Day 9 Day 12 Day 16Day 21 Cmpd 5-2 >30 >30 >30 >30 >30 24.2 14.5 Sofosbuvir >30 >30 >3012.1 6.8  2.7  2.3 INX-189 >30 4.2 1.8 1.8 1.3 <<1   <<1  

Example 33. Metabolic Studies

The metabolism of compounds 25, 27 and 5-2, at a concentration of 10 μM,were investigated in fresh primary cultures of human, dog and mousehepatocytes. Plated hepatocytes from humans (XenoTech, mixed gender,pooled from 10 donors), male Beagle dog (BioreclamationIVT), and maleICR/CD-1 mice (BioreclamationIVT, 8 donors) in 6-well plates withmatrigel overlay were incubated in singlet with 10 μM TA. After 2, 4, 6,8 or 24 hr, intracellular levels of nucleotide prodrugs and theirpotential metabolites (prodrugs, monophosphates, triphosphates andnucleosides) were quantitated by LC-MS/MS. Concentrations below thelower limit of quantitation (1.5 μmol/10⁶ cells for prodrugs,monophosphates and nucleosides and 12 μmol/10⁶ cells for triphosphates)were extrapolated from the standard curves.

The compound β-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-guanine triphosphateis the predominant metabolite of compounds 25, 27 and 5-2 observed incultured human hepatocytes and is a potent inhibitor of the HCV (gt1b)NS5B polymerase, with an IC₅₀ of 0.15 μM.

FIG. 8 shows the predominant Compound 25 metabolites in humanhepatocytes.

FIG. 9 shows the predominant Compound 27 metabolites in humanhepatocytes.

FIG. 10 shows the predominant Compound 5-2 metabolites in humanhepatocytes.

FIG. 11 illustrates the activation pathways for Compounds 25, 27 and5-2. As can be seen, Compounds 25, 27 and 5-2 are converted to theircorresponding monophosphate analogs which are subsequently metabolizedto a common MP analog; β-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-guaninemonophosphate (Compound 61). The monophosphate is then stepwisephosphorylated to the active triphosphate:β-D-2′-deoxy-2′-α-fluoro-2′-β-methyl-guanine triphosphate (Compound 62).

Example 34. Controls

INX-189 (INX-08189BMS-986094) and sofosbuvir were used as controls inthe Examples above.

The two most potent nucleotide prodrugs, Compounds 25 and 27,demonstrated excellent selectivity, with CC₅₀ values greater than 100 μMin Huh-7 cells, human bone marrow stem cells and human cardiomyocytes.No inhibition of human DNA polymerase, α, β or γ, no activity againstother RNA or DNA viruses, and no toxicity in all host cell lines wasobserved at concentrations up to 100 μM.

Table 7 is a table illustrating the compounds tested in a HCV RepliconAssay along with the EC₅₀/EC₉₅ (μM) and CC₅₀ (μM) results.

TABLE 7 Replicon Assay Results for Compounds Tested. Fold increase HCVHCV in activity Relicon Replicon compared Cmpd EC₅₀/EC₉₅ CC₅₀ to parentNo. Structure (μM) (μM) nucleoside

6.7 >100

 2.1/9.04 >100 3  4

15.7 >100  5

0.026/0.124 >100 >600 5-1

0.0551/0.282  >100 >280 5-2

0.004/0.028 >100 >3,900  6

10.7 >100  7

0.0121/0.071  >100 >890  8

5.56 >100  9

0.0091/0.054  >100 >600 15

>100 >100 16

0.576/3.69  >100 17

11.5/65.4 >100 18

0.048/0.219 90.0 19

7.47 >100 20

0.073/0.315 >100 25

0.004/0.019 >100 >2,600 26

0.0351/0.057  >100 27

0.005/0.025 >100 >1,100 28

0.014/0.076 >100 41

0.508/25.1  21.8 42

4.18/20.4 >100 43

6.43/24.7 21.6 45

 0.16/0.876 0.68 46

0.224/0.961 >100 47

0.338/1.72  1.68 61

62

0.052/0.310 >100

0.045/0.259 >100

The β-D-2′-D-2′-α-fluoro-2′-β-C-substituted-2-modified-N⁶-substitutedpurine nucleotides described herein exhibit significant activity againstthe HCV virus. Compounds according to the present invention are assayedfor desired relative activity using well-known and conventional assaysfound in the literature.

For example, anti-HCV activity and cytotoxicity of the compounds may bemeasured in the HCV subgenomic RNA replicon assay system in Huh7 ETcells. (See, Korba, et al., Antiviral Research 2008, 77, 56). Theresults can be summarized in comparison to a positive control,2′-C-Me-cytosine {2′-C-Me-C}(Pierra, et al., Journal of MedicinalChemistry 2006, 49, 6614.

Another in-vitro assay for anti-hepatitis C virus activity is describedin U.S. Pat. No. 7,718,790 by Stuyver, et al., and assigned toPharmasset, Inc.

This specification has been described with reference to embodiments ofthe invention. Given the teaching herein, one of ordinary skill in theart will be able to modify the invention for a desired purpose and suchvariations are considered within the scope of the invention.

We claim:
 1. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein the compound isat least 90% free of the opposite phosphorus S-enantiomer.
 2. Thecompound of claim 1, wherein the compound is at least 98% free of theopposite phosphorus S-enantiomer.
 3. The compound of claim 1, whereinthe compound is at least 99% free of the opposite phosphorusS-enantiomer.
 4. A pharmaceutical composition comprising a compound ofthe formula

or a pharmaceutically acceptable salt thereof, wherein the compound isat least 90% free of the opposite phosphorus S-enantiomer.
 5. Thepharmaceutical composition of claim 4, wherein the compound is at least98% free of the opposite phosphorus S-enantiomer.
 6. The pharmaceuticalcomposition of claim 4, wherein the compound is at least 99% free of theopposite phosphorus S-enantiomer.
 7. The pharmaceutical composition ofclaim 4, in an oral dosage form.
 8. The pharmaceutical composition ofclaim 7, wherein the oral dosage form is a solid dosage form.
 9. Thepharmaceutical composition of claim 8, wherein the solid dosage form isa tablet.
 10. The pharmaceutical composition of claim 8, wherein thesolid dosage form is a capsule.
 11. The pharmaceutical composition ofclaim 4, wherein the oral dosage form is a liquid dosage form.
 12. Thepharmaceutical composition of claim 11, wherein the liquid dosage formis a suspension or solution.
 13. The pharmaceutical composition of claim4, in an intravenous formulation.
 14. The pharmaceutical composition ofclaim 4, in a parenteral formulation.